Functional module for regulating drought response of plants and application thereof

Abstract

The invention discloses a functional module for regulating drought response of plants and application of the functional module. The functional module comprises TaPYR10 and downstream signal channel members thereof. According to the application disclosed by the invention, a new molecular basis is provided for the drought tolerance of wheat by revealing the effects of the TaPYR10 and downstream partners (TaPP2C30, TaSnRK2.10 and TaNF-YC1) thereof in drought signal transduction. Functional analysis of the genes shows that the genes play a key role in response of plants to drought stress, and stress resistance of the plants can be improved by adjusting related physiological processes (such as stomatal movement, synthesis of osmotic regulation substances and cell active oxygen steady state).

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a functional module for regulating plant drought response and its application. Background Art

[0002] Drought stress, caused by reduced precipitation and limited water supply, is one of the most damaging abiotic stresses that adversely affect plant growth, development, and crop yield formation. In recent years, it is predicted that global climate change will exacerbate the degree of water stress in crops in the future and intensify the negative impact of drought on crop production. It is predicted that by the end of this century, drought will affect up to 60% of the current wheat planting area. Therefore, using crop varieties with improved drought tolerance has been a long-term focus of modern sustainable agriculture.

[0003] Numerous studies have shown that drought stress has a significant impact on physiological, biochemical, and molecular processes that affect water relations at the cellular, tissue, and organ levels. For major crops, drought leads to a significant decrease in relative water content, chlorophyll content, and plant productivity. Therefore, plants have evolved various strategies to mitigate the negative effects of drought, enhancing their osmotic adjustment ability by accumulating osmoregulatory substances (such as proline, soluble sugars, betaine, organic acids, free amino acids, and other organic compounds) to maintain sufficient water supply. Plants can also activate antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) to improve the reactive oxygen species scavenging ability and maintain the reactive oxygen species homeostasis. In addition, plant hormones such as auxin (IAA), gibberellin (GA), zeatin riboside (ZR), abscisic acid (ABA), and jasmonic acid (JA) act synergistically to mediate plant drought response.

[0004] Abscisic acid (ABA) is the main stress-responsive hormone produced by plants in response to drought. It affects multiple physiological processes throughout the plant life cycle, including seed dormancy and germination, root growth, stomatal guard cell swelling, senescence, and fruit ripening. The ABA-mediated stress response is achieved through a specific ABA signaling pathway in which plant cells perceive the ABA signal and convert it into various physiological and biochemical reactions through signal transduction processes. When plants are exposed to drought conditions, members of the ABA receptor family (i.e., PYR / PYL / RCAR) play a role in perceiving the drought signal in the environment and triggering ABA signal transduction through a protein phosphorylation mechanism. It has been confirmed that members of the PYR / PYL / RCAR, type 2C protein phosphatase (PP2C) proteins, and SNF1-related protein kinase 2 (SnRK2) family members are the core signaling components of the ABA-dependent pathway triggered by drought. Recent studies have established a working model of the ABA signaling pathway. Under conditions of sufficient water and low ABA levels, the activity of SnRK2 members is inhibited by physical interaction with type A PP2C (PP2C-A) proteins, which dephosphorylate multiple serine / threonine residues located in the activation loop. This inhibition suppresses the physiological, biochemical, and molecular processes involved in the plant drought response. In addition, under drought conditions, the ABA concentration in tissues increases and then binds to PYR / PYL / RCAR proteins, enabling the ABA receptor to interact with PP2C proteins, resulting in the release of SnRK2 from the inhibition by PP2C, thus activating downstream targets such as ABA-responsive element binding factors (ABFs), ABA-responsive element binding proteins (AREBs), and transcription factor members such as NAC, MYC, and MYB. Therefore, the ABA receptor-mediated signaling pathway is a key regulator of the plant drought response.

[0005] Recent studies have identified and characterized members of the NF-Y family, as well as members of the PP2C and SnRK2 families, in the model plant Arabidopsis thaliana. Functional characterization of NF-Y genes has shown that they play a key role as mediators of the plant drought stress response, as they regulate biological processes related to drought stress response. Among the NF-Y genes in maize, ZmNF-YA1 and ZmNF-YB16 have been shown to confer better drought adaptability to plants by enhancing the transcription of osmotic stress-responsive genes and improving the water use efficiency of plants under drought stress. Members of the ABA receptor family PYR / PYL / RCAR are located downstream of the NF-Y transcription factors, and TaPYL10 has been shown to upregulate the expression of ABA biosynthesis genes (including ZEP1, NCED1, NCED2, NCED3, and NCED4), which helps to maintain cell water content, membrane stability, chlorophyll biosynthesis, reduce the levels of reactive oxygen species (such as H2O2), and improve plant productivity under drought conditions. In Arabidopsis thaliana, ten members of the SnRK2 family have been identified, and expression analysis has shown that all SnRK2 members in Arabidopsis thaliana, except SnRK2.9, are activated by osmotic stress. Functional characterization has confirmed that they are involved in transducing osmotic stress signals through the ABA-dependent pathway. In addition, type A PP2C proteins in Arabidopsis thaliana play a crucial role as negative regulators in the ABA signaling pathway. Therefore, different members of the ABA receptor family and their downstream partners form signaling pathways that mainly regulate stress defense biological processes through the ABA-dependent pathway, thereby contributing to plant drought tolerance.

[0006] Wheat is an important crop widely cultivated worldwide. With the intensification of global warming, soil drought has become increasingly severe, having a negative impact on wheat yields. Although ABA receptors and their downstream partners (such as PP2C proteins and SnRK2 members) have been extensively characterized in model plant species and some crop species, in common wheat, the functional characterization of members of the ABA receptor family, their downstream partners in the PP2C and SnRK2 families, and members of the transcription factors involved in plant drought defense remains limited. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a functional module for regulating plant drought response and its application.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0009] A functional module for regulating plant drought response, comprising TaPYR10 and its downstream signaling pathway members.

[0010] More preferably, the downstream signaling pathway members include TaPP2C30, TaSnRK2.10, and TaNF-YC1.

[0011] Method for cultivating drought-resistant transgenic plants, gene editing of TaPYR10 gene and its downstream signaling pathway members, and cultivating transgenic plants with drought-resistant characteristics.

[0012] Further preferably, specifically overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibiting or reducing or silencing or knocking out TaPP2C30 gene.

[0013] Kit for regulating the drought response ability of plants, the kit contains molecular biology elements capable of regulating the expression level of specific genes; the specific genes are genes related to regulating the drought response ability of plants.

[0014] Further preferably, the molecular biology elements are a combination of elements for overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out TaPP2C30 gene.

[0015] A method for enhancing the drought stress tolerance of plants, overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out TaPP2C30 gene to enhance the drought stress tolerance of plants.

[0016] A method for increasing the yield of plants, overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out TaPP2C30 gene to enhance the drought stress tolerance of the corresponding plants, thereby increasing the yield of the corresponding plants.

[0017] According to the above functional module or the above method or the above kit or the above method or the above method, the plant is wheat.

[0018] Use of a functional module including TaPYR10 and its downstream signaling pathway members, by analyzing the expression levels of TaPYR10 and its downstream signaling modules to evaluate the drought response ability of plants, screening, pre-screening, identifying, and assisting in identifying wheat germplasm resources with strong drought resistance ability, specifically screening, pre-screening, identifying, and assisting in identifying wheat germplasm overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibiting or reducing or silencing or knocking out TaPP2C30 gene.

[0019] A method for enhancing the drought stress tolerance of wheat, introducing multiple copies of cis-element DRE and MYB and MYC TF protein binding sites into the wheat genome by gene editing technology, and constructing a system that can overexpress TaPYR10 under drought and ABA signal stimulation to enhance the drought stress tolerance of wheat.

[0020] An SNP locus related to proline content, biomass and yield in wheat, characterized in that: the SNP locus is located on chromosome 1 of wheat, and the physical distance of the SNP locus in the wheat genome version Triticum aestivumiwgsc_refseqv1.0 is Chr1A_346681497. The 4 SNP loci in the 0RF of TaPYR10 correspond to the bases at positions 211, 224, 374, and 410 of the sequence shown in SEQ ID NO.1 respectively. When these loci are homozygous for G / G, T / T, G / G, A / A in sequence, the corresponding genotype is Hap1. When the loci are homozygous for T / T, A / A, C / C, G / G, the corresponding genotype is Hap2; the 5 SNP loci in the promoter region correspond to the bases at positions 1680, 1684, 1685, 1737, and 1902 counted from the end of the sequence shown in SEQ ID NO.9 respectively. When these loci are homozygous for T / T, A / A, C / C, A / A, G / G in sequence, the corresponding genotype is Hap1. When the loci are homozygous for C / C, G / G, T / T, G / G, T / T, the corresponding genotype is Hap2; the proline content, biomass and yield are as follows: wheat homozygous for the genotype Hap1 is greater than or candidate greater than wheat homozygous for the genotype Hap2.

[0021] A primer combination, characterized in that it is used to detect the single nucleotide polymorphism of the following SNP loci in the wheat genome. The physical distance of the SNP locus in the wheat genome version Triticum aestivum iwgsc_refseqv1.0 is Chr1A_346681497. The 4 SNP loci in the 0RF of TaPYR10 correspond to the 211th, 224th, 374th, and 410th bases of the sequence shown in SEQ ID NO.1 respectively. When these loci are homozygous for G / G, T / T, G / G, A / A in sequence, the corresponding genotype is Hap1. When the loci are homozygous for T / T, A / A, C / C, G / G, the corresponding genotype is Hap2. The 5 SNP loci in the promoter region correspond to the 1680th, 1684th, 1685th, 1737th, and 1902nd bases from the end of the sequence shown in SEQ ID NO.9 respectively. When these loci are homozygous for T / T, A / A, C / C, A / A, G / G in sequence, the corresponding genotype is Hap1. When the loci are homozygous for C / C, G / G, T / T, G / G, T / T, the corresponding genotype is Hap2. The proline content, biomass, and yield are as follows: The wheat homozygous for the genotype Hap1 is greater than or potentially greater than the wheat homozygous for the genotype Hap2. The primer combination is a primer pair composed of SEQ ID NO.244 to SEQ ID NO.279 in the sequence listing. This primer combination is used to detect the SNP loci described in claim 1.

[0022] Develop Kasp molecular markers related to wheat drought resistance according to the described SNP loci.

[0023] A method for cultivating drought-tolerant and high-yield wheat varieties, including the following steps: In the early breeding stage, for the wheat hybrid offspring population, detect the transcript abundances of the TaPYR10 gene and stress defense genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. According to the detection results, preferentially select individuals from the hybrid offspring population whose transcript abundances of the TaPYR10 gene and the stress defense genes are all higher than a preset threshold. The preset threshold is determined by collecting a large number of wheat varieties with different drought tolerance and yield performances, testing the transcript abundances of each gene in the upper leaves at the mid-filling stage and the yield data at maturity under field drought conditions, and through statistical analysis. Cultivate the selected individuals to obtain wheat varieties or lines with high drought tolerance and high yield characteristics, and greatly improve the breeding efficiency and shorten the breeding cycle through this method.

[0024] The beneficial effects of adopting the above technical solutions are as follows: In this study, we focused on characterizing the ABA receptor gene TaPYR10 in wheat to identify its downstream partners and functionally characterize its ABA core signaling module in mediating plant drought responses. Our research results provide new insights into understanding the mechanism of the ABA signaling pathway in plant drought responses and may promote the development of drought-tolerant varieties of common wheat through molecular breeding;

[0025] The present invention provides a new molecular basis for the drought tolerance of wheat by revealing the roles of TaPYR10 and its downstream partners (TaPP2C30, TaSnRK2.10, and TaNF-YC1) in drought signal transduction. Functional analysis of these genes indicates that they play a key role in plant responses to drought stress and can improve plant stress resistance by regulating related physiological processes (such as stomatal movement, synthesis of osmoregulatory substances, and cellular reactive oxygen species homeostasis);

[0026] Through the analysis of cis-regulatory elements in the TaPYR10 promoter, the present study reveals that its expression is precisely regulated by drought and ABA signals, providing new insights into understanding the gene regulation mechanism in plants under environmental stress, offering new targets for genetic engineering and breeding, and improving breeding efficiency;

[0027] By constructing transgenic wheat lines, the present invention verifies the functions of TaPYR10 and its downstream partners in drought responses, providing genetic resources for subsequent crop improvement, especially in enhancing wheat drought tolerance, with important application prospects. Brief Description of the Drawings

[0028] Figure 1 It is a schematic diagram for the characterization of the TaPYR10 protein, where A is the alignment result between the TaPYR10 protein and its counterparts in various plant species; B is the simulated three-dimensional structure of the TaPYR10 protein; C is the comparison of fluorescence signals between TaPYR10-GFP and GFP in N. benthamiana and wheat protoplasts; in A and B, the conserved domains designated by I to IX are highlighted, and in C, the arrow points to the nucleus;

[0029] Figure 2 It is a schematic diagram of the phylogenetic relationship of TaPYR10 and its plant homologs at the nucleic acid level;

[0030] Figure 3Schematic diagram of the expression pattern of TaPYR10 under drought and ABA treatments. Among them, Figures A - B are schematic diagrams of the expression pattern of TaPYR10 in roots and leaves under drought treatment (Figure A) and ABA treatment (Figure B); in Figure A, 0, 1%, 5%, 10%, and 15% represent the concentrations of PEG (w / v), 1h, 3h, 9h, and 27h represent the time after drought treatment (10% PEG, w / v), R1h, R3h, R9h, and R27h represent the time of recovery treatment after 27h of drought challenge, and 0h represents the time point before drought treatment; in Figure B, 0, 0.5 μmol, 1 μmol, 1.5 μmol, and 2 μmol represent the concentrations of exogenous ABA, and 1h, 3h, 9h, and 27h represent the time after ABA treatment (1.5 μmol ABA); Figure C is a schematic diagram of the binary expression cassette integrated with the TaPYR10 promoter; Figure D is a schematic diagram showing cis - regulatory elements related to osmotic stress, namely MYB (CAACAG, CAACCA, and CCGTTG), DRE (GCCGAC), and MYC (TCTCTTA) located in the TaPYR10 promoter; Figures E - F are schematic diagrams of the staining results (Figure E) and GUS activity (Figure F) of the reporter gene GUS driven by the truncated TaPYR10 promoter in plants. D1 to D5 represent different regions of the TaPYR10 promoter (454bp, 745bp, 1378bp, 1666bp, and the full - length 1764bp). In Figure F, the data are represented as mean ± standard deviation (n = 3), and Student's t - test was used to test the statistical significance between transgenic lines under the same growth conditions (*p < 0.05);

[0031] Figure 4Schematic diagram of the experimental results of the protein interaction between TaPYR10 and its downstream partners; among them, Figure A is a schematic diagram showing the predicted interaction between TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 proteins; Figures B-D are schematic diagrams of the yeast two-hybrid experimental results, indicating the interactions between TaPYR10 and TaPP2C30 (Figure B), TaPP2C30 and TaSnRK2.10 (Figure C), and TaSnRK2.10 and TaNF-YC1 (Figure D); Figure E is a schematic diagram of the BiFC experimental results to verify the interaction between TaPYR10 and its downstream partners; Figures F-H are schematic diagrams of the co-immunoprecipitation experimental results, showing the interactions between TaPYR10 and TaPP2C30 (Figure F), TaPP2C30 and TaSnRK2.10 (Figure G), and TaSnRK2.10 and TaNF-YC1 (Figure H); Figures I-K are schematic diagrams of the in vitro pull-down experimental results, indicating the protein interactions between TaPYR10 and TaPP2C30 (Figure I), TaPP2C30 and TaSnRK2.10 (Figure J), and TaSnRK2.10 and TaNF-YC1 (Figure K);

[0032] Figure 5Schematic diagram of experimental results of protein fragments interacting with TaPYR10 and its downstream partners; in which Figure A is a schematic diagram of each fragment of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins, and these fragments were used in yeast two-hybrid and BiFC experiments; Figure B is a schematic diagram of the yeast two-hybrid experimental results, indicating the contributions of different fragments of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins to protein interactions; Figure C is a schematic diagram of the BiFC experimental results, verifying the fragments involved in the interactions between TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins; in Figures B and C, TaPYR10I1 represents the middle fragment of TaPYR10 (amino acids 44-192), TaPP2C30I1 represents the middle fragment of TaPP2C30 (amino acids 74-317), TaSnRK2.10I1 represents the middle fragment of TaSnRK2.10 (amino acids 22-278), and TaNF-YC1N1 represents the N-terminal of TaNF-YC1 (amino acids 81-255); Figures D-F are schematic diagrams of the co-immunoprecipitation experimental results, and the results indicate different fragments involved in the protein interaction between TaPYR10 (TaPYR10I1) and TaPP2C30 (TaPP2C30I1) (Figure D), the interaction between TaPP2C30 (TaPP2C30I1) and TaSnRK2.10 (TaSnRK2.10I1) (Figure E), and the interaction between TaSnRK2.10 (TaSnRK2.10I1) and TaNF-YC1 (TaNF-YC1N1) (Figure F); Figures G-I are schematic diagrams of the in vitro pull-down experimental results, verifying the fragments involved in the interactions between TaPYR10 and its downstream partners, including TaPYR10I1 and TaPP2C30I1 (Figure G), TaPP2C30I1 and TaSnRK2.10I1 (Figure H), and TaSnRK2.10I1 and TaNF-YC1N1 (Figure I);

[0033] Figure 6 Schematic diagram of experimental results of evaluating the interactions between TaPYR10 and PP2C, SnRK2 and NF-YC family proteins by yeast two-hybrid method, in which Figure A is the yeast two-hybrid experimental results between TaPYR10 and PP2C family members; Figure B is the yeast two-hybrid experimental results between TaPP2C30 and SnRK2 family members; Figure C is the yeast two-hybrid experimental results between TaSnRK2.10 and NF-YC family members;

[0034] Figure 7Schematic diagram of the expression levels of target genes of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 in transgenic wheat lines; among them, Figures A-D are the expression levels of target genes in the following transgenic lines: A is the TaPYR10 transgenic line; B is the TaPP2C30 transgenic line; C is the TaSnRK2.10 transgenic line; D is the TaNF-YC1 transgenic line; the expression values were normalized by Tatubulin and Taactin, and the expression levels of both were set to 1; the data are mean ± standard deviation (n = 3); different lowercase letters indicate significant differences between the transgenic lines and the wild type (WT) (Tukey test, P < 0.05);

[0035] Figure 8 Schematic diagram of the phenotypes and growth characteristics of transgenic lines of TaPYR10 and its downstream partner genes under drought treatment; among them, Figures A-D are schematic diagrams of the phenotypic results during the middle grain filling period under drought treatment; Figures E-H are schematic diagrams of the plant biomass results during the middle grain filling period when growing in the field; Figures I-L are schematic diagrams of the leaf area results of plants during the middle grain filling period under field conditions; Figures M-P are schematic diagrams of the grain weight results of plants cultivated under field conditions; Figures Q-T are schematic diagrams of the yield results of plants growing under field conditions; in Figures E-T, the soil moisture content under normal conditions is 70%-75% of the field capacity; in Figures A-T, under drought treatment, the soil humidity is 55%-60% of the field capacity; the data are expressed as mean ± standard deviation (n = 3), and Student's t-test was used to test the statistical significance between the transgenic lines and the WT under the same growth conditions (*p < 0.05);

[0036] Figure 9 Schematic diagram of the plant phenotypes of transgenic lines (overexpressed or knocked down) of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 growing under normal field conditions;

[0037] Figure 10Schematic diagrams of the phenotypes and plant biomass of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 transgenic lines under exogenous ABA treatment; in which, Figure A is the schematic diagram of the phenotypic results of TaPYR10 transgenic lines; Figure B is the schematic diagram of the phenotypic results of TaPP2C30 transgenic lines; Figure C is the schematic diagram of the phenotypic results of TaSnRK2.10 transgenic lines; Figure D is the schematic diagram of the phenotypic results of TaNF-YC1 transgenic lines; Figure E is the schematic diagram of the plant biomass results of TaPYR10 transgenic lines; Figure F is the schematic diagram of the plant biomass results of TaPP2C30 transgenic lines; Figure G is the schematic diagram of the plant biomass results of TaSnRK2.10 transgenic lines; Figure H is the schematic diagram of the plant biomass results of TaNF-YC1 transgenic lines; exogenous ABA (1 μmol) was sprayed at the second leaf stage; in Figures E-H, the data are presented as mean ± standard deviation (n = 3). Student's t-test was used to test the statistical significance between transgenic lines and WT (*p < 0.05 and **p < 0.01);

[0038] Figure 11 Schematic diagrams of the osmotic stress-related physiological characteristics of TaPYR10 and its downstream partner transgenic lines; in which, Figure A is the schematic diagram of the stomatal characteristics results under drought stress; Figures B-E are the schematic diagrams of the stomatal closure rate results during 2-hour drought treatment; Figures F-I are the schematic diagrams of the leaf water loss results during 2-hour water dehydration; Figures J-M are the schematic diagrams of the proline content results; Figures N-Q are the schematic diagrams of the soluble sugar content results; in Figures B-Q, the data are presented as mean ± standard deviation (n = 3), and Student's t-test was used to test the statistical significance between transgenic lines and WT under similar conditions (*p < 0.05);

[0039] Figure 12 Schematic diagrams of the photosynthetic parameter results of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 transgenic lines under drought treatment; in which, Figures A-D are the schematic diagrams of the net photosynthetic rate (Pn) results; Figures E-H are the schematic diagrams of the actual quantum efficiency of photosystem II (ΦPSII) results; Figures I-L are the schematic diagrams of the stomatal conductance (Gs) results; Figures M-P are the non-photochemical quenching coefficient (NPQ); the data are presented as mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences between transgenic lines at the same growth stage (Tukey test, P < 0.05);

[0040] Figure 13Schematic diagrams of ROS-related indexes and root phenotypes of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment; in which, Figures A-D are schematic diagrams of superoxide anion content evaluated by nitroblue tetrazolium (NBT) staining; Figures E-H are schematic diagrams of H2O2 content evaluated by 3,3-diaminobenzidine (DAB) staining; Figures I-L are schematic diagrams of superoxide dismutase (SOD) activity results; Figures M-P are schematic diagrams of catalase (CAT) activity results; Figures Q-T are schematic diagrams of crude enzyme (POD) activity results; Figures U-X are schematic diagrams of root phenotype results; in Figures I-T, the data are presented as mean ± standard deviation (n = 3), and Student's t-test was used to test the statistical significance between WT and transgenic lines (*p<0.05);

[0041] Figure 14 Schematic diagrams of superoxide anion and H2O2 content results of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment; in which, Figures A-D are schematic diagrams of superoxide anion content in transgenic lines evaluated by nitroblue tetrazolium (NBT) staining; Figures E-H are schematic diagrams of H2O2 content in transgenic lines evaluated by 3,3-diaminobenzidine (DAB) staining;

[0042] Figure 15 Schematic diagrams of MDA content and root characteristics of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment; Figures A-D are schematic diagrams of MDA content results; Figures E-H are schematic diagrams of root biomass results; Figures I-L are schematic diagrams of root volume results; the data are presented as mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences between transgenic lines under the same growth conditions (Tukey test, P<0.05);

[0043] Figure 16Schematic diagram of the results of the expression, yeast one-hybrid, and transcriptional activation characteristics of genes related to the stress response. Figures A-E are schematic diagrams of the expression of the SLAC1 gene (Figure A), genes related to proline accumulation (Figure B), SOD gene (Figure C), CAT gene (Figure D), and PIN gene (Figure E) in TaNF-YC1 overexpression lines under drought treatment; Figure F is a schematic diagram of cis-acting elements in the promoters of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. ABRE, ABA response element (CACGTG, ACGTG, AACCCGG, GACACGTGGC, CGCACGTGTC); MYB, drought response element (CAACCA, CAACAG, TAACCA); Figure G is a schematic diagram of the results of yeast one-hybrid experiments, showing that the hosts transformed with pGADT7-TaNF-YC1 and pHIS2-TaSLAC1-3pro, pGADT7-TaNF-YC1 and pHIS2-TaP5CR1pro, pGADT7-TaNF-YC1 and pHIS2-TaSOD4pro, pGADT7-TaNF-YC1 and pHIS2-TaCAT2pro, and pGADT7-TaNF-YC1 and pHIS2-TaPIN6pro grew normally on triple dropout media (SD / -Leu-Trp-His) plates containing 45 mM 3-AT; Figures H-L are schematic diagrams of the luciferase signals detected after co-transforming the effector CaMV35SPro::TaNF-YC1 and the reporter genes TaSAC1-3Pro::LUC (Figure H), TaP5CR1Pro::LUC (Figure I), TaSOD4Pro::LUC (Figure J), TaCAT2Pro::LUC (Figure K), and TaPIN6Pro::LUC (Figure L) in N. benthamiana epidermal cells; Figures M-Q are schematic diagrams of the LUC activity results in leaves co-transformed with the effector and reporter gene combinations mentioned in Figures H-L; The data in Figures M-Q are presented as mean ± standard deviation (n = 3), and Student's t-test was used to test the statistical significance of the LUC activities caused by the effector / reporter genes (*p < 0.05);

[0044] Figure 17Schematic diagram of the physiological indexes and root morphological characteristics of gene knockdown lines under drought treatment. Among them, Figures A - C are schematic diagrams of the stomatal morphology (Figure A), stomatal aperture (Figure B), and transpiration rate (Figure C) of TaSLAC1 - 3 knockdown lines within 1 hour during drought treatment. AntiSLAC1 - 3A and AntiSLAC1 - 3B are TaSLAC1 - 3 knockdown lines; Figures D - F are schematic diagrams of the phenotypes (Figure D), plant biomass (Figure E), and proline content (Figure F) of TaP5CR1 knockdown lines during the seedling stage. AntiP5CR1 - 1 and AntiP5CR1 - 2 are TaP5CR1 knockdown lines; Figures G - I are schematic diagrams of the phenotypes (Figure G), superoxide dismutase (SOD) activity (Figure H), and histochemical staining to show the accumulated superoxide anions (Figure I) of TaSOD4 knockdown lines during the seedling stage. AntiSOD4 - 2 and AntiSOD4 - 3 are TaSOD4 knockdown lines; Figures J - L are schematic diagrams of the phenotypes (Figure J), catalase (CAT) activity (Figure K), and histochemical staining to show the accumulated H2O2 (Figure L) of TaCAT2 knockdown lines during the seedling stage. AntiCAT2 - 2 and AntiCAT2 - 3 are TaCAT2 knockdown lines; Figures M - O are schematic diagrams of the root morphology (Figure M), root biomass (Figure N), and root volume (Figure O) of TaPIN6 knockdown lines cultivated under field conditions. AntiPIN6 - 1 and AntiPIN6 - 2 are TaPIN6 knockdown lines; Data are mean ± standard deviation (n = 3), and Student's t - test was used to test the statistical significance between transgenic lines and wild - type under the same growth conditions (*p < 0.05).;

[0045] Figure 18 Schematic diagram of the contents of superoxide anions and H2O2 in TaSOD4 and TaCAT2 knockdown lines under drought treatment;

[0046] Figure 19Schematic diagram of the transcriptional characteristics of TaPYR10 and its downstream stress defense genes and crop yield characterization results under field drought conditions in wheat varieties. In Figure A, the expression level results of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 in the tested wheat varieties are shown; Figure B shows the yield results in the tested wheat varieties; Figures C-H are schematic diagrams of the regression analysis results of yield with TaPYR10 transcript (Figure C), yield with TaSLAC1-3 transcript (Figure D), yield with TaP5CR1 transcript (Figure E), yield with TaSOD4 transcript (Figure F), yield with TaCAT2 transcript (Figure G), and yield with TaPIN6 transcript (Figure H), respectively; in Figures A-B, 45 tested wheat varieties are shown on the left and are simplified according to the registered names.

[0047] Figure 20 Schematic diagram of the effects of TaPYR10 haplotypes on proline, plant biomass, and yield traits. In Figure A, the alignment results of TaPYR10 haplotypes Hap1 and Hap2 and their deduced amino acid sequences are shown, and the polymorphic sites shown in red are used to develop TaPYR10 haplotype-specific markers; Figure B is a display diagram of genotyping a group of wheat varieties using TaPYR10 haplotype-specific markers; Figure C is a haplotype heatmap clustering based on genes related to TaPYR10; Figures D, E, and F are schematic diagrams of the results of yield, plant biomass, and proline, respectively, between wheat varieties with contrasting haplotypes Hap1 and Hap2.

[0048] Figure 21 Schematic diagram of the working model of TaPYR10 and its downstream partners in mediating plant drought responses. Detailed implementation manners

[0049] The following examples illustrate the present invention in detail. All kinds of raw materials and various equipment used in the present invention are conventional commercially available products and can be directly obtained through market purchase. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0050] It should be understood that when used in the specification of the present application and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements, and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0051] It should also be understood that the term "and / or" as used in the specification of this application and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0052] Reference to "one embodiment" or "some embodiments" or the like described in the specification of this application means that a specific feature, structure, or characteristic described in connection with that embodiment is included in one or more embodiments of this application. Thus, statements such as "in one embodiment", "in some embodiments", "in other some embodiments", "in still other embodiments", etc. that appear in different places in this specification do not necessarily all refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "comprising", "including", "having" and their variants all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0053] In addition, in the description of the specification of this application and the appended claims, the terms "first", "second", "third", etc. are only used for distinguishing descriptions and cannot be understood as indicating or implying relative importance.

[0054] Hereinafter, the technical solution of the present invention will be clearly and completely described in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0055] Embodiment 1, Materials and Methods

[0056] (1) Plant materials and growth conditions: Wheat (Shimai 22) seedlings grown hydroponically in a growth chamber were used to analyze the expression of TaPYR10 under drought and ABA treatments. Seedlings of TaPYR10 (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), its downstream partner TaPP2C30 (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4), TaSnRK2.10 (nucleotide sequence as shown in SEQ ID NO.5, amino acid sequence as shown in SEQ ID NO.6), TaNF-YC1 (nucleotide sequence as shown in SEQ ID NO.7, amino acid sequence as shown in SEQ ID NO.8), and transgenic lines of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 regulated by TaNF-YC1 were cultured in plastic pots containing soil (half vermiculite and half fertile soil) in a growth chamber to determine the function of the genes in mediating plant drought and ABA responses.

[0057] The wheat seedlings were cultured under the following growth conditions: 18 - 22 °C, 16-hour light / 8-hour dark cycle, with supplementary light provided by high-pressure sodium lamps (Powertone SON-TAGRO 400W; Philips Electronic UK Ltd, Farnborough, UK).

[0058] The above transgenic lines and a core wheat cultivar population consisting of 45 cultivars were evaluated for growth traits and gene expression levels in a field experiment conducted at the experimental station of the Cereal and Oil Crops Research Institute, Hebei Academy of Agriculture and Forestry Sciences, Shijiazhuang, Hebei Province, China (37°56′24.62″ N, 114°42′46.96″ E) during the 2022 - 2023 and 2023 - 2024 growing seasons.

[0059] Table 1 Information on the core wheat cultivar population of 45 cultivars participating in the field experiment

[0060]

[0061]

[0062]

[0063] (2) Molecular characterization analysis

[0064] The molecular characteristics of the wheat ABA receptor (PYR) family member TaPYR10 (GenBank accession number TraesCS1A02G191700) were characterized as follows: The open reading frame (ORF) of TaPYR10 and its 2-kb-long promoter region (nucleotide sequence shown in SEQ ID NO. 9) were retrieved from the Ensembl Plants website (www.http: / / plants.ensembl.org / index.html). The conserved domains similar to other PYR counterparts of the TaPYR10 protein were defined. By performing a BLASTn search analysis on the GenBank database of the National Center for Biotechnology Information (NCBI, https: / / www.blast.ncbi.nlm.nih.gov / Blast.cgi), the phylogenetic relationship between TaPYR10 and its homologous genes in different plant species was obtained, and their association was established using the MegAlign algorithm in DNAStar software (https: / / www.dnastar.com). The ClustalW alignment analysis of the TaPYR10 protein and its plant counterparts was performed using MEGA11 software (https: / / www.mega.com). The three-dimensional structure of the TaPYR10 protein was modeled using the online tool SWISS-MODEL algorithm (https: / / swissmodel.expasy.org / interactive), and the relevant prediction programs were used as recommended.

[0065] (3) Protein subcellular localization analysis

[0066] The subcellular localization of the TaPYR10 protein was determined by detecting the signal of TaPYR10-GFP (green fluorescent protein, GFP) in Nicotiana benthamiana epidermal cells. For this purpose, the ORF of TaPYR10 was amplified using gene-specific primers based on reverse transcription polymerase chain reaction (RT-PCR) and was frame-integrated with the reporter gene (GFP) into the binary vector pCAMBIA3300 under the control of the CaMV35S promoter. Then, the resulting expression cassette TaPYR10-GFP was transformed into Agrobacterium (strain EHA105) using the conventional heat shock method. The positive transformants were used for the transient transformation of Nicotiana benthamiana epidermal cells using the Agrobacterium-mediated transformation method (Li et al., 2012). At 48 hours after transformation, the GFP signals in the cells containing the TaPYR10-GFP fusion construct and the cells integrated with the empty vector were observed using a fluorescence microscope.

[0067] Table 2 Gene-specific primers

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] (4) Expression analysis of TaPYR10

[0074] Seedlings of three-leaf stage wheat (cultivar Shimai 22) cultured in modified Murashige-Skoog (MS) solution supplemented with polyethylene glycol (PEG) and exogenous ABA were used to analyze the expression pattern of TaPYR10 in response to drought and ABA signals. The PEG concentrations used were 0, 1, 5, 10, and 15% (w / v), corresponding to osmotic potentials of 0, -0.35, -0.81, -1.08, and -1.27 MPa in the solution, respectively. The ABA levels in the MS solution were 0.5, 1.0, 1.5, and 2.0 μmol. In addition, seedlings collected at different time points (0 h before treatment and 1, 3, 9, and 27 h after treatment) under drought (10% PEG) and ABA (1 μmol) treatments were used to characterize the temporal expression pattern of TaPYR10 in response to the above signal conditions. TaPYR10 transcripts in roots and leaves were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific primers (Table 2). Two constitutive genes, tubulin (Tatubulin) and actin (Taactin) in wheat, were used as internal references to normalize the target transcripts.

[0075] (5) β-Glucuronidase (GUS) assay

[0076] In transgenic wheat lines carrying truncated promoter fragments, GUS histochemical staining and GUS activity driven by various cis-acting regulatory elements predicted to be in the TaPYR10 promoter by the Plant Cis-Acting Regulatory Element Database (PlantCARE, http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) were determined. For this purpose, the full-length promoter (1764 bp) and a set of promoter regions of TaPYR10, namely 454 bp, 745 bp, 1378 bp, and 1666 bp, which contain various cis-acting regulatory elements (i.e., DRE and MYB, MYC recognition sites) affecting gene transcription and osmotic stress response (Table 3), were amplified from wheat genomic DNA (Shimai 22) using gene-specific primers (Table 2). Transgenic wheat (Shimai 22) lines carrying these cassettes (i.e., TaPYR10 promoter fragments and the reporter gene (β-glucuronidase, GUS)) were obtained using the Agrobacterium-mediated transformation method (Kumar et al., 2019). After 6 hours of drought treatment (10% PEG), GUS histochemical staining and activity assessment were performed on representative leaves of T2 transgenic lines (Houde et al., 2020).

[0077] Table 3 Information table of drought-related cis-acting elements and stress response-related genes in the TaPYR10 promoter

[0078]

[0079]

[0080]

[0081] (6) Analysis of the expression pattern of TaPYR10 downstream partners

[0082] Expression analysis was performed on TaSLAC1 family members that regulate stomatal movement, family members involved in proline biosynthesis, the SOD family that affects superoxide dismutase activity, the CAT family that affects catalase activity, and the PIN-FORMED (PIN) family genes that control auxin transport and root architecture behavior in TaNF-YC1 transgenic lines under drought stress. The SLAC1 genes analyzed included TaSLAC1-1 to TaSLAC1-6, the proline biosynthesis genes included TaP5CS1, TaP5CS2, TaP5CR1, TaProDH1, and TaP5CDH1, the SOD genes included TaSOD1 to TaSOD6, the CAT genes included TaCAT1 to TaCAT6, and the PIN genes included TaPIN1 to TaPIN6. The root tissues of three-leaf stage seedlings cultured in modified MS solution (10% PEG) were evaluated for the target expression levels after 27 hours of treatment. Similar to the qRT-PCR operation performed on TaPYR10 under drought stress, gene-specific primers (Table 2) were used to detect the transcripts of the above genes. Under field drought conditions (the soil water potential in the plow layer was -1.05 mPa), the expression patterns of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, TaPIN6, and TaPYR10 in 45 tested wheat varieties (Table 1) were evaluated at the mid-filling stage. Among them, transgenic and wild-type plants were conventionally planted in field plots (1 m long and 0.3 m wide), while wheat varieties were planted in plots (8 m long and 4 m wide), with three replicates. Drought stress was established through conventional deficit irrigation management (irrigation was carried out before sowing and at the jointing stage). Representative flag leaves of the varieties were collected using gene-specific primers (Table 2) and the target transcripts were evaluated based on qRT-PCR.

[0083] (7) Yeast two-hybrid assay

[0084] Use the yeast two-hybrid assay to identify the partners of the ABA signaling module involved by TaPYR10, namely PP2C proteins, SnRK2 kinases, and NF-Y transcription factors. The PP2C family members used in the experiment included TaPP2C3, TaPP2C5, TaPP2C7, TaPP2C10, and TaPP2C30; the SnRK2 family members included TaSnRK2.5 to TaSnRK2.10; and the NF-Y family members included TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC5, and TaNF-YC8 (Table 2). For this purpose, the ORF of TaPYR10 was amplified by RT-PCR and integrated into the expression vector pGADT7 to generate the cassette pGADT7-TaPYR10 (bait). Meanwhile, the ORFs of the PP2C genes were amplified separately and inserted into the expression vector pGBKT7 as prey. The yeast host (strain AH109) containing the bait (TaPYR10) and prey (each TaPP2C protein) was cultured on selective solid medium supplemented with exogenous ABA (1 μmol) to initiate protein-protein interactions. Using a similar strategy, the ORFs of the PP2C members that interacted with TaPYR10 were amplified by RT-PCR as bait, while the ORFs of the SnRK2 members were amplified and inserted into the expression vector pGBKT7 as prey, respectively. Similarly, the SnRK2 members that interacted with the above PP2C proteins were amplified and inserted into the vector pGADT7 as bait, while the ORFs of the NF-YC members were amplified and inserted into the expression vector pGBKT7 as prey, respectively. The positive transformants containing the bait and prey were cultured on the selective medium SD / -Leu / -Trp lacking specific amino acids for 3 days at 30 °C. Fragments of TaPYR10 [i.e., TaPYR10-N1 (amino acids 1 to 70), TaPYR10-I1 (amino acids 71 to 140), and TaPYR10-C1 (amino acids 141 to 211)], TaPP2C30 [TaPP2C30-N1 (amino acids 1 to 73), TaPP2C30-I1 (amino acids 74 to 317), and TaPP2C30-C1 (amino acids 318 to 406)], TaSnRK2.10 [TaSnRK2.10-N1 (amino acids 1 to 90), TaSnRK2.10-I1 (amino acids 91 to 260), and TaSnRK2.10-C1 (amino acids 261 to 394)], and TaNF-YC1 [TaNF-YC1-N1 (amino acids 1 to 80) and TaNF-YC1-C1 (amino acids 81 to 255)] were amplified by RT-PCR using gene-specific primers (Table 2). Then they were used to construct bait or prey, and yeast two-hybrid assays similar to the above were performed.

[0085] (8) Bimolecular fluorescence complementation (BiFC) assay

[0086] The BiFC assay was performed to verify the in vivo protein interaction between TaPYR10 and its downstream partners. For this purpose, the ORFs of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 were amplified using RT-PCR and gene-specific primers (Table 2) and were frame-integrated with fragments of the reporter gene YFP (yellow fluorescent protein gene) at the C-terminus (pSPYCE(M)-TaPYR10 and pSPYCE(M)-TaSnRK2.10) or N-terminus (pSPYNE(R)-TaPP2C30 and pSPYNE(R)-TaNF-YC1) as described previously (Shen et al., 2011). Expression cassettes combinations, namely pSPYCE(M)-TaPYR10 and pSPYNE(R)-TaPP2C30, pSPYNE(R)-TaPP2C30 and pSPYCE(M)-TaSnRK2.10, pSPYCE(M)-TaSnRK2.10 and pSPYNE(R)-TaNF-YC1 were used for genetic co-transformation of Agrobacterium (strain EHA105) and further transiently transformed young leaves of Nicotiana benthamiana (Kumar et al., 2019). Two days after infiltration, YFP signals in transgenic leaves were detected using a fluorescence microscope (Olympus FV10-ASW, Japan) according to the manufacturer's instructions. While detecting the YFP signals, nuclear targets were identified using the nuclear marker DAPI (Solarbio, Beijing, China) as described previously (Sun et al., 2013). Various fragments of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 identical to those in the yeast two-hybrid assay above were amplified and the BiFC assay was performed using gene-specific primers (Table 2). YFP signals from transformed young leaves of N. benthamiana were detected in a similar manner as above.

[0087] (9) Co-Immunoprecipitation (Co-IP) assay

[0088] The open reading frames (ORFs) of TaPYR10 and its downstream partners TaPP2C30, TaSnRK2.10, and TaNF-YC1, as well as the cDNA sequences encoding different fragments of the above signal members (TaPYR10I1, TaPP2C30I1, TaSnRK2.10I1, and TaNF-YC1N1), were amplified using RT-PCR and gene-specific primers (Table 2). Then they were used to construct expression cassettes, namely MET-TaPYR10 / TaPYR10I1-GFP, MET-TaPP2C30 / TaPP2C30I1-GFP, MET-TaSnRK2.10 / TaSnRK2.10I1-GFP, and MET-TaNF-YC1 / TaNF-YC1N1-GFP, and used to transform the Agrobacterium strain EHA105. The positive transformants were used for transient transformation of Nicotiana benthamiana leaves (Zhao et al., 2022). Briefly, about 0.3 g of leaves were collected 24 h after infiltration and ground into powder using 1 mL of buffer A (50 mM hEPES-NaOH pH 7.5, 150 mM NaCl, 5% glycerol, 0.5% Triton X-100, 0.1% (v / v) 3-mercapto-1,2-propanediol, one Complete Mini protease inhibitor cocktail tablet per 10 mL (Shangon, Shanghai, China)). The resulting solution was centrifuged twice at 1000 g for 10 min at 4 °C to obtain the protein fraction. They were further mixed with an equal volume of 2× sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [(Laemmli sample buffer (BIO-RAD, California, USA) 950 μL / mL, 3-mercapto-1,2-propanediol 50 μL / mL)] and denatured at 95 °C for 5 min, then stored at -30 °C. The proteins were incubated with GFP-trap agarose beads (Chromotek, Planegg, Germany) at 4 °C for protein binding. After washing the beads 4 - 6 times with buffer A, the proteins were eluted with 1× SDS-PAGE sample buffer (50% 2× SDS-PAGE sample buffer, 50% 1× PBS). Immunoprecipitation experiments were performed using a 12.5% SDS-PAGE gel (e-PAGEL) (ATTO, Tokyo, Japan).

[0089] (10) In vitro pull-down assay

[0090] An in vitro pull-down experiment was performed to verify the protein interactions shown in the above Co-IP experiment. The ORFs of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1, as well as the cDNA sequences encoding different fragments of the above signal members, were amplified by RT-PCR using gene-specific primers (Table 2) and cloned into the protein expression vector pGEX-4T-1 with a glutathione S-transferase (GST) tag, respectively. Meanwhile, the ORFs of the downstream partners of TaPYR10 and the cDNA sequences encoding different protein fragments (TaPP2C30I1, TaSnRK2.10I1, and TaNF-YC1N1) were amplified and inserted into the expression vector pMAL-c2X with a maltose-binding protein (MBP) tag, respectively. The plasmids were routinely transformed into Escherichia coli BL21(DE3) strains for protein expression. Positive transformants were collected at 4 °C and then resuspended in GST pull-down buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM MgCl2, and 0.5% Lgepal CA-630) and sonicated in an ice bath. The resulting supernatant was separated and subjected to SDS-PAGE to detect the protein components. Equal amounts of TaPYR10-GST and its downstream proteins were incubated in 500 μl of GST pull-down buffer at 4 °C for 6 h, and then 100 μl of GST beads (GE healthcare, New Jersey, USA) was added to the mixture and incubated at 4 °C for 2 h. The pulled-down proteins were detected using an anti-MBP antibody (New England Biolabs, Los Angeles, USA) and an anti-GST antibody (Abcam, Cambridge, UK) according to the manufacturer's instructions.

[0091] (11) Construction of transgenic wheat lines

[0092] Construct transgenic wheat lines of TaPYR10 and its downstream partners TaPP2C30, TaSnRK2.10, TaNF-YC1, and TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 regulated by TaNF-YC1 to study their roles in mediating drought responses. Briefly, the ORFs of the above genes were amplified in the forward or reverse direction by RT-PCR using gene-specific primers (Table 2). The amplified products were separately integrated into the NcoI / BastEII sites of the binary vector pCAMBIA3301 under the control of the CaMV35S promoter. The resulting expression cassettes were used for genetic transformation of Agrobacterium tumefaciens (strain EHA105), which was then further transformed into common wheat (cultivar Shimai 22) (Kumar et al., 2019). The target transcripts in the transgenic lines were evaluated by qRT-PCR using gene-specific primers (Table 2). Representative lines with overexpression and knockdown expression of each gene and the wild type (WT) were selected in the T3 generation, grown under normal conditions, and subjected to drought or ABA treatment.

[0093] (12) Establishment of drought and ABA treatments

[0094] Drought and ABA treatments were established for transgenic lines of TaPYR10 and its downstream partners to characterize the functions of the genes in mediating plant stress responses. For drought treatment, the transgenic lines and wild type (WT) were grown under growth chamber or field conditions. For growth chamber cultivation, transgenic and WT seedlings were planted in plastic pots filled with a soil mixture (half vermiculite and half fertile soil) and watered daily to maintain a relative soil moisture of 70 - 75%. At the three-leaf stage, they were divided into two groups: one group was grown under normal watering conditions, and the other group was subjected to drought treatment by restricting water supply to maintain a relative soil moisture of 55 - 60% (measured using a soil water potential meter TRS-IIN, Zhejiang, China). For field planting, transgenic and WT plants were conventionally planted in field plots (each 1 m long and 0.3 m wide), with three replicates. Two irrigations (before sowing and at the jointing stage) were carried out, which was considered the conventional deficit irrigation management for local winter wheat (the soil water potential in the plough layer was -1.05 to -1.22 mPa during the filling stage). ABA treatment was established by externally spraying 1 μmol ABA on seedlings or plants two weeks before the experiment for the determination of growth, physiological, and biochemical indices.

[0095] (13) Evaluation of growth traits and physiological indices

[0096] After drought treatment, the growth traits and drought-related physiological indices of transgenic lines and WT plants were evaluated. The growth traits evaluated included phenotype and plant biomass. Among them, photographs were taken using a digital camera to record the phenotype; plant biomass was obtained by drying in an oven from five representative seedlings (grown in a growth chamber) or twenty plants (grown in the field) using conventional methods. At maturity, the phenotype of the seeds was recorded, and the grain weight was evaluated after the seeds were air-dried. The plot yield was based on the air-dried grain weight of 0.5 m 2 plants. The physiological indices evaluated included those related to the osmotic stress response, such as the content of osmotic adjustment substances, stomatal closure rate (SCR), water loss rate of detached leaves (WLR), and reactive oxygen species-related parameters, using the upper expanded leaves as samples. Among them, the contents of the osmotic adjustment substances proline and soluble sugar were evaluated (Zhao et al., 2024). The value of the stomatal closure rate (SCR) was determined according to the ratio of the stomatal width observed at different drought time points [0.25, 0.5, and 1 hour during 1-hour drought treatment (10% PEG)] to that at 0 hour (Zhao et al., 2022). The value of the water loss rate of detached leaves (WLR) was determined according to the reduction in fresh weight recorded at different time points (0.5, 1, and 3 hours) relative to 0 hour during the drought treatment (starting from placing the leaves on a clean bench). The reactive oxygen species-related indices, such as the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), the content of malondialdehyde (MDA), and the accumulation of reactive oxygen species (i.e., superoxide anion and H2O2) were evaluated as follows: the enzyme activities and MDA content were determined (Huang et al., 2010); the amounts of superoxide anion and H2O2 were evaluated using histochemical staining methods, i.e., nitroblue tetrazolium (NBT) staining for superoxide anion and 3,3'-diaminobenzidine (DAB) staining for H2O2 (Zhao et al., 2024).

[0097] (14) Transcription activation experiment

[0098] Transcription activation experiments were conducted on five differentially expressed genes, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6, in TaNF-YC1 transgenic lines under drought stress in the Nicotiana benthamiana expression system. For this purpose, the ORF of TaNF-YC1 was amplified using RT-PCR and gene-specific primers (Table 2) and integrated into the pGreenII 62-SK vector to generate the effector cassette CaMV35Spro::TaNF-YC1. Meanwhile, the promoter regions (2 kb in length) of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 were amplified using wheat (Shimai 22) genomic DNA as a template and gene-specific primers (Table 2) and integrated into the pGreenII0800-LUC vector, respectively, to generate the reporter constructs TaSLAC1-3pro::Luc, TaP5CR2::Luc, TaSOD4::Luc, TaCAT2::Luc, and TaPIN6pro::Luc. The recombinant binary cassettes were co-transformed into the Agrobacterium tumefaciens strain EHA105 at a ratio of 1:1 (v:v), with combinations of CaMV35Spro::TaNF-YC1 and TaSLAC1-3pro::LUC, CaMV35Spro::TaNF-YC1 and TaP5CR1::LUC, CaMV35Spro::TaNF-YC1 and TaSOD4pro::LUC, CaMV35Spro::TaNF-YC1 and TaCAT2pro::LUC, and CaMV35Spro::TaNF-YC1 and TaPIN6pro::LUC. The positive transformants were further used for transient transformation of Nicotiana benthamiana epidermal cells (Wang et al., 2020). After 48 hours of transformation and spraying with 100 mM luciferin, the reporter luciferin signal in the transiently transformed cells was evaluated using a charge-coupled device imaging device (NightOWLII LB983 combined with Indigo software) according to the manufacturer's instructions.

[0099] (15) Yeast one-hybrid assay

[0100] Yeast one-hybrid assays were performed according to the previous method (Zhang et al., 2024) to verify the transcriptional activation of TaNF-YC1 on its target genes, namely TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. For this purpose, the ORF of TaPYR10 was amplified by RT-PCR using gene-specific primers (Table 2) and integrated into the pGADT7 vector (Clontech) at the EcoRI and KpnI sites to generate the fusion protein pGADT7-TaPYR10 (as prey). Meanwhile, the promoter regions of its target genes were inserted into the XhoI and KpnI sites of the pHIS2 reporter vector (Clontech) to generate the fusion proteins pHIS2-TaSLAC1-3, pHIS2-TaP5CR1, pHIS2-TaSOD4, pHIS2-TaCAT2, and pHIS2-TaPIN6 (as baits). Combinations consisting of the prey and each bait plasmid were co-transformed into the yeast strain (EGY48). Positive transformants were identified after culturing on solid selective growth medium lacking SD-Trp / -Ura for 3 days.

[0101] (16) Statistical analysis

[0102] The means of gene transcripts, growth traits, plant biomass, and osmotic stress-related physiological indices were from the results of three replicates. The statistical analysis system software (SAS Corporation, Cary, NC, USA) was used to determine the standard error of the means and evaluate the statistical significance of the traits. Analysis of variance (ANOVA) was used to evaluate the regression analysis results between plant biomass and the expression levels of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 in the cultivar population under field drought conditions. Student's t-test (critical value P≤0.05) was used to determine the statistical significance analysis of transgenic lines and WT in plant morphological traits and physiological indices. The software called TBtools was used to draw a heatmap showing the expression levels of TaPYR10 and stress-responsive genes as well as the wheat cultivar yields in the field experiment.

[0103] Example 2. Results

[0104] (1) Molecular characteristics of TaPYR10

[0105] The cDNA length of TaPYR10 was 633 bp, encoding a polypeptide consisting of 211 amino acids with a molecular weight of 22.62 kDa and an isoelectric point (pI) of 4.97.

[0106] At the amino acid level, TaPYR10 has a high similarity with its counterparts from Triticum dicoccoides, Aegilops tauschii, Paspalum hallii, Panicum miliaceum, Zea mays, Brachypodium distachyon, Sorghum bicolor, and Avena brachyantha; they all contain a conserved PYR_PYL_PCAR domain consisting of nine blade units ( Figure 1 A).

[0107] At the nucleic acid level, TaPYR10 has a high identity with homologous genes in different plant species and has the highest similarity with genes from Paspalum halli, Panicum virgatum, Setaria viridis, Oryza japonica, Zea mays, Avena brachyantha, Hordeum vulgare, Aegilops tauschii, and Triticum dicoccoides ( Figure 2 ).

[0108] These results indicate that TaPYR10 has a similar evolutionary pathway to its plant counterparts. Based on three-dimensional (3-D) structure analysis, it was found that TaPYR10 contains a six-bladed β-propeller domain and three α-helices, which together form nine structural sites that constitute the PYR_PYL_PCAR domain. In addition, TaPYR10 contains an A8S-type binding site for abscisic acid. These binding sites are involved in binding ABA and interacting with downstream proteins ( Figure 1 B).

[0109] In the model plant Nicotiana benthamiana and wheat protoplasts, transiently transformed cells expressing the TaPYR10-GFP fusion protein showed that the GFP signal was localized to the nucleus ( Figure 1 C), indicating that the TaPYR10 protein is localized to the nucleus after endoplasmic reticulum (ER) sorting and is involved in biological functions.

[0110] (2) Expression sensitivity of TaPYR10 to drought signals

[0111] Analysis of the TaPYR10 transcript revealed its expression changes in response to drought and ABA signals. Under conditions of increasing drought, the expression levels of TaPYR10 in both roots and leaves were significantly upregulated and reached the highest level at 15% PEG treatment ( Figure 3A). In addition, during the 27-hour drought treatment, the TaPYR10 transcripts in both tissues gradually increased and returned to normal levels after 27 hours of recovery conditions. Figure 3 A). A similar TaPYR10 expression pattern was also observed in plant tissues treated with exogenous ABA; the transcripts of this wheat PYR gene were shown to be induced by ABA signals in an ABA concentration- and time-dependent manner. Figure 3 B). These findings indicate that TaPYR10 is highly sensitive to drought and ABA signals at the transcriptional level. Online prediction analysis showed that the TaPYR10 promoter contains a set of potential cis-acting regulatory elements related to gene transcription under osmotic stress, including the conserved motifs TATA box and CAAT box that regulate gene transcription, the element DRE responsive to osmotic stress, and the binding sites of transcription factors MYB and MYC that mediate gene transcription under stress. Figure 3 C-D). These cis-regulatory elements are considered to play an important role in regulating TaPYR10 transcription under drought conditions. Under drought conditions, compared with the control group (non-stress conditions), GUS staining and GUS activity in leaves transformed with the full-length promoter (D5, 1764 bp) and truncated TaPYR10 promoter fragments D1 (454 bp), D2 (745 bp), D3 (1378 bp), and D4 (1666 bp) were enhanced. Figure 3 D-F). In addition, as the promoter fragment was extended, GUS staining and activity in the leaves gradually increased. Figure 3 D-F). These results suggest that the cis-element DRE and the binding sites of MYB and MYC transcription factors coordinately regulate the transcription of TaPYR10 in response to drought and ABA signals.

[0112] (3) TaPYR10 forms an ABA core signaling module with TaPP2C30, TaSnRK2.10, and TaNF-YC1

[0113] Online prediction analysis indicated that the TaPYR10 protein can interact with the PP2C member TaPP2C30, which can in turn interact with the SnRK2 member TaSnRK2.10. Similarly, the above SnRK2 member interacts with the NF-YC member TaNF-YC1. Figure 4 A). Based on the prediction results, an experimental analysis of the interaction between TaPYR10 and its putative downstream partners was conducted. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and in vitro pull-down assays were used to study the role of TaPYR10 in establishing the ABA signaling module. In the yeast two-hybrid assay, the TaPYR10 protein (bait) expressed in yeast cells specifically interacted with the PP2C member TaPP2C30 (prey) expressed in the yeast host.Figure 4 B). Similarly, it was found that TaPP2C30 protein interacted specifically with TaSnRK2.10, and the latter interacted with TaNF-YC1 in the same experimental system ( Figure 4 C-D). The protein-protein interactions identified from yeast two-hybrid experiments were further confirmed by BiFC, and clear YFP signals were detected in Nicotiana benthamiana epidermal cells co-transformed with TaPYR10-nYFP and TaPP2C30-cYFP, TaPP2C30-cYFP and TaSnRK2.10-nYFP, and TaSnRK2.10-nYFP and TaNF-YC1-cYFP ( Figure 4 E). Co-IP and in vitro pull-down analyses of the above ABA signaling components verified the protein interaction results obtained from yeast two-hybrid and BiFC experiments. Among them, Co-IP analysis showed that TaPYR10 could co-immunoprecipitate with TaPP2C30, TaPP2C30 could co-immunoprecipitate with TaSnRK2.10, and TaSnRK2.10 could co-immunoprecipitate with TaNF-YC1 ( Figure 4 F-H). In vitro pull-down analysis confirmed the protein interactions between the above ABA signaling partners ( Figure 4 I-K). These findings indicate that TaPYR10 forms an ABA core signaling module TaPYR10 / TaPP2C30 / TaSnRK2.10 / TaNF-YC1 with its downstream partners TaPP2C30, TaSnRK2.10 and TaNF-YC1, which plays a crucial role in transducing stress-induced ABA signals and influencing drought responses.

[0114] (4) Protein fragments of the interaction between TaPYR10 and its downstream partners

[0115] To study the different regions involved in the protein-protein interactions among TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1, their N-terminal and C-terminal fragments, as well as the middle fragments of TaPYR10 and TaPP2C30 ( Figure 5 A) were expressed and analyzed using protein interaction experiments. In yeast two-hybrid experiments, positive yeast transformants were identified in cells co-expressing the middle fragment of TaPYR10 (TaPYR10-I1, amino acids 71 to 140, containing the CL3-CL5 loop and the PYR / PYL / PCAR conserved domain) and the middle fragment of TaPP2C30 (TaPP2C30-I1, amino acids 74 to 317, containing the PP2C conserved activation domain) ( Figure 5B). Similarly, yeast host cells co-expressing the middle fragment of TaPP2C30-I1 and TaSnRK2.10 (TaSnRK2.10-I1, amino acids 91 to 260, containing the conserved serine / threonine kinase activation site), and yeast host cells co-expressing TaSnRK2.10-I1 and the N-terminus of TaNF-YC1 (TaNF-YC1-N1, amino acids 1 to 80, containing the nuclear localization signal) showed complementary growth ability on the selective medium SD / -Leu / -Trp lacking amino acids ( Figure 5 B). In the yeast two-hybrid assay, no interactions were detected between TaPYR10 and other PP2C proteins, TaPP2C30 and other SnRK2 members, and TaSnRK2.10 and other NF-YC proteins, except for the downstream partners of TaPYR10 described previously ( Figure 6 ). BiFC assays confirmed the interactions between these fragments in TaPYR10 and its downstream signaling partners. High-sensitivity detection of the reporter gene YFP signal was achieved in Nicotiana benthamiana epidermal cells co-transformed with TaPYR10-I1-nYFP / TaPP2C30-I1-cYFP, TaPP2C30-I1-cYFP / TaSnRK2.10-I1-nYFP, and TaSnRK2.10-I1-nYFP / TaNF-YC1-N1-cYFP ( Figure 5 C). Further Co-IP and in vitro pull-down analyses confirmed the above interaction processes obtained from the yeast two-hybrid and BiFC assays. Both assays showed that TaPYR10I1 co-immunoprecipitated with TaPP2C30I1, TaPP2C30I1 co-immunoprecipitated with TaSnRK2.10I1, and TaSnRK2.10I1 co-immunoprecipitated with TaNF-YC1N1 ( Figure 5 D-I). These findings indicate that different protein regions in TaPYR10 and its downstream partners contribute to protein-protein interactions among ABA signaling pathway members.

[0116] (5) Transgenic lines of TaPYR10 and its downstream partner genes alter plant drought responses

[0117] Transgenic wheat lines of TaPYR10 and its downstream partner genes were constructed to characterize the function of the genes in mediating plant drought response. Under normal growth conditions, the lines of TaPYR10 (Sen 1, Sen 2, Anti 1, and Anti 2), TaPP2C30 (Sen2, Sen 3, Anti 1, and Anti 2), TaSnRK2.10 (Sen 1, Sen2, Anti 2, and Anti 3), and TaNF-YC1 (Sen2, Sen 3, Anti 2, and Anti 3) had significant changes in the expression of target genes ( Figure 7 ), but were similar to wild-type (WT) plants in terms of growth traits, i.e., similar phenotypes, biomass, leaf area, grain weight, and yield ( Figure 8 A-T, Figure 9 ). Under drought conditions, the growth characteristics of all transgenic lines changed. The lines overexpressing TaPYR10 (Sen 1 and Sen2), TaSnRK2.10 (Sen 1 and Sen 2), and TaNF-YC1 (Sen 2 and Sen 3), and the lines with TaPP2C30 knockdown (Anti 1 and Anti 2) had increased phenotypes ( Figure 8 A-D), increased biomass ( Figure 8 E-H), increased leaf area ( Figure 8 I-L), increased grain weight ( Figure 8 M-P), and increased yield ( Figure 8 Q-T) compared with wild-type (WT) plants. In contrast, the lines with TaPYR10 (Anti 1 and Anti2), TaSnRK2.10 (Anti 2 and Anti 3), and TaNF-YC1 (Anti 2 and Anti 3) knockdown, and the lines overexpressing TaPP2C30 (Sen 2 and Sen 3) had deteriorated phenotypes ( Figure 8 A-D), and the plant biomass, leaf area, grain weight, and yield were reduced compared with WT plants ( Figure 8 E-T). The growth trait performances of the above transgenic lines under exogenous ABA treatment were similar to those under the above drought conditions ( Figure 10 ). The changes in the growth traits of these transgenic lines indicate that TaPYR10 and its signaling pathway genes play a key role in regulating plant drought and ABA responses.

[0118] (6) Physiological characteristics related to osmotic stress of TaPYR10 and its downstream partner transgenic lines

[0119] The physiological characteristics and indices related to osmotic stress response in TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 lines were evaluated, including stomatal closure rate (SCR), water loss rate (WLR) of detached leaves, contents of osmoregulatory substances proline and soluble sugar, photosynthetic parameters, reactive oxygen species-related indices, and root morphological characteristics. Within 1 h of drought treatment, the stomatal closure rate increased in TaPP2C30 knockdown (Anti 1 and Anti 2) and TaPYR10 (Sen 1 and Sen 2), TaSnRK2.10 (Sen 1 and Sen2), and TaNF-YC1 (Sen2 and Sen 3) overexpressing lines compared with the WT ( Figure 11 A-E). During 2 h of leaf dehydration, the water loss rate of detached leaves in these lines showed a decreasing trend relative to WT plants ( Figure 11 F-I). These lines also showed increased proline ( Figure 11 J-M) and soluble sugar ( Figure 11 N-Q) contents, enhanced photosynthetic capacity (increased net photosynthetic rate Pn, stomatal conductance Gs, and actual quantum efficiency of photosystem II ΦPSII, decreased non-photochemical quenching coefficient NPQ) ( Figure 12 ), and improved reactive oxygen species-related indices, i.e., decreased superoxide anion and H2O2 contents ( Figure 13 A-H, Figure 14 A-H), increased activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) ( Figure 13 I-T), and decreased malondialdehyde (MDA) accumulation ( Figure 15 A-D). The root system architecture (RSA) characteristics of these transgenic lines, i.e., root morphology ( Figure 13 U-X), root biomass ( Figure 15 E-H), and root volume ( Figure 15 I-L), were consistent with their roles in mediating plant drought response. In contrast, TaPP2C30 overexpressing (Sen2 and Sen 3) and TaPYR10 (Anti 1 and Anti 2), TaSnRK2.10 (Anti 2 and Anti 3), and TaNF-YC1 (Anti 2 and Anti 3) knockdown transgenic lines showed opposite effects under drought treatment, i.e., decreased stomatal closure rate, increased water loss rate of detached leaves, decreased proline and soluble sugar contents, decreased photosynthetic capacity, deteriorated reactive oxygen species homeostasis-related indices ( Figure 11 A-Q, 13A-T, 15A-D), and alleviated root system architecture-related characteristics ( Figure 13 U-X, 15E-L). Overall, the TaPYR10 signaling module plays an important role in mediating physiological processes related to plant stress response.

[0120] (7) TaNF-YC1 transcriptionally activates the expression of osmotic stress-responsive genes

[0121] Expression analysis was performed on a set of genes, including the SLAC1 family genes (TaSLAC1-1 to TaSLAC1-6) that regulate stomatal movement, genes related to proline accumulation (TaP5CS1, TaP5CS2, TaP5CR1, TaProDH1, TaP5CDH1), the superoxide dismutase (SOD) family (TaSOD1 to TaSOD6) and catalase (CAT) family (TaCAT1 to TaCAT6) genes that mediate antioxidant enzyme activity, and the PIN-FORMED (PIN) family genes (TaPIN1 to TaPIN6) that regulate root architecture establishment. These genes were analyzed in TaNF-YC1 transgenic lines after drought and exogenous ABA treatment. Among the genes tested, the expression levels of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 were altered in TaNF-YC1 lines relative to WT plants (higher expression in Sen 2 and Sen 3, lower expression levels in Anti 2 and Anti 3)( Figure 16 A-E). In contrast, the transcripts of other tested genes did not change in TaNF-YC1 transgenic lines treated with drought compared to WT( Figure 16 A-E). These results indicate that the differentially expressed genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 are affected by TaNF-YC1 at the transcriptional level. Yeast one-hybrid assays were performed to determine the protein / DNA interactions between TaNF-YC1 and the promoters of the differentially expressed genes. Figure 16 F is a schematic diagram showing the distribution of cis-elements responsive to drought (MYB) and ABA (ABRE) in stress-responsive genes. As expected, the hosts co-transformed with pGADT7-TaNF-YC1 and pHIS2-TaSLAC1-3Pro, pGADT7-TaNF-YC1 and pHIS2-TaP5CR1Pro, pGADT7-TaNF-YC1 and pHIS2-TaSOD4pro, pGADT7-TaNF-YC1 and pHIS2-TaCAT2Pro, and pGADT7-TaNF-YC1 and pHIS2-TaPIN4Pro grew normally on plates of triple-deficient medium (SD / -Leu-Trp-His) containing 45 mmol L -1 3-AT Figure 16G), indicating that TaNF-YC1 interacts with the promoters of stress-responsive genes. Transcription activation experiments were conducted in Nicotiana benthamiana to verify the regulation of TaNF-YC1 on the above stress defense genes. Strong reporter gene LUC signals were detected in tobacco epidermal cells transiently co-transformed with the following plasmid combinations: effector cassette CaMV35SPro::TaNF-YC1 and reporter cassette TaSLAC1-3Pro::LUC( Figure 16 H, M); effector cassette CaMV35SPro::TaNF-YC1 and reporter cassette TaP5CR1Pro::LUC( Figure 16 I, N); effector cassette CaMV35SPro::TaNF-YC1 and reporter cassette TaSOD4Pro::LUC( Figure 16 J, O); effector cassette CaMV35SPro::TaNF-YC1 and reporter cassette TaCAT2Pro::LUC( Figure 16 K, P); and effector cassette CaMV35SPro::TaNF-YC1 and reporter cassette TaPIN6Pro::LUC( Figure 16 L, Q). These results together confirmed that the transcription of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 is regulated by TaNF-YC1 and affects osmotic stress-related physiological processes under drought conditions.

[0122] (8) Positive regulation of differentially expressed osmotic stress-responsive genes on adaptation to drought stress

[0123] Transgenic analysis of the differentially expressed stress-responsive genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 was performed to characterize their roles in mediating drought responses. Under drought treatment, compared with WT plants, the TaSLAC1-3 knockdown lines (AntiSLAC1-3A and AntiSLAC1-3B) showed deteriorated phenotypes, reduced stomatal closure rates, and increased transpiration rates( Figure 17 A-C); the TaP5CR1 expression knockdown lines (AntiP5CR1-1 and AntiP5CR1-2) grew slowly, had reduced biomass, and reduced proline content( Figure 17 D-F); TaSOD4 knockdown (AntiSOD4-2 and AntiSOD4-3) led to phenotypic changes, reduced SOD activity, and increased superoxide anion accumulation( Figure 17 G-I, Figure 18 A); the TaCAT2 knockdown lines (AntiCAT2-2 and AntiCAT1-3) had poor phenotypes, reduced CAT activity, and increased H2O2 accumulation( Figure 17 J-L, Figure 18(B); The root growth of TaPIN6 knockdown lines (AntiPIN6-1 and AntiPIN6-2) was significantly reduced, root biomass decreased, and root volume was smaller ( Figure 17 (M - O). In the absence of drought treatment, there were no significant differences in plant phenotypes and root growth between these transgenic lines and WT plants. These results verified the positive role of osmotic stress-related genes in drought adaptation at the transcriptional level of TaNF-YC1, a member of the TaPYR10 module.

[0124] (9) The transcript abundances of TaPYR10 and its downstream stress defense genes in wheat varieties were highly correlated with yield under field drought conditions

[0125] In a set of 45 wheat varieties with different drought tolerances in terms of plant yield performance, the expression levels of TaPYR10 and stress-responsive genes (TaSLAC1 - 3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) were detected. Under field drought conditions, the transcripts of these genes showed significant differences in the upper leaves at the mid-filling stage ( Figure 19 (A). Similarly, under field drought conditions, there were also huge differences in the yields of the tested wheat varieties at maturity ( Figure 19 (B). Regression analysis was performed to characterize the relationship between gene transcripts and yield in wheat varieties under drought stress. The results showed that yield was significantly positively correlated with the expression levels of TaPYR10 and four stress-responsive genes ( Figure 19 (C - H). These findings indicate that TaPYR10 and different stress defense genes act synergistically at the transcriptional level to regulate plant drought responses and contribute to plant drought adaptation ability.

[0126] (10) Haplotype variation behavior of TaPYR10

[0127] To identify sequence variations of TaPYR10, single nucleotide polymorphism (SNP) sites in the open reading frame (ORF) and SNP sites in the promoter region were detected by sequencing the ORF and flanking regions (i.e., 2 kb upstream of the start codon) of 45 wheat varieties. Four SNPs in the ORF of TaPYR10 and five SNPs in the promoter region formed two major haplotypes, namely TaPYR10-Hap1 and TaPYR10-Hap2 ( Figure 20 A). Based on the above results, a competitive allele-specific PCR (KASP) marker called TaPYR10-KASPA523T was developed and used to genotype the 45 elite wheat varieties tested ( Figure 20B). The expression levels of TaPYR10 and stress-responsive genes (TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) were characterized. Heatmap analysis showed that wheat varieties classified as TaPYR10-Hap1 had higher expression levels than those classified as TaPYR10-Hap2 ( Figure 20 C), resulting in plants with higher proline content, plant biomass, and yield ( Figure 20 D-F). These results together indicate that TaPYR10-Hap1 has been under positive selection in modern wheat breeding programs.

[0128] Example 3. Conclusion

[0129] (1) The response of TaPYR10 to drought signals is related to specific cis-elements in the promoter

[0130] The expression of ABA signaling genes is strictly regulated at the transcriptional level. For example, due to the increase in ABA levels in plant cells, the transcripts of PYR family genes are altered in response to drought, salt, and ABA stresses (Antoni et al., 2012; Li et al., 2018). Drought and exogenous ABA treatments can also significantly induce the expression of PP2C genes regulated by different transcription factors such as the MYB, ABF, and NF-Y families (Wang and Qin, 2017; Zhang et al., 2017). Studies have shown that a heterotrimer formed by members of the nuclear factor YC subunit (NF-YC) family transcription factors GmNF-YC14, GmNF-YA16, and GmNF-YB2 in soybean activates the abscisic acid (ABA) signaling pathway mediated by GmPYR1, thereby regulating the stress tolerance of soybean (Yu et al., 2021). In this study, our expression analysis of TaPYR10 showed that it responds to drought and ABA signals, and its expression levels in roots and leaves depend on the degree and duration of stress. These findings indicate that TaPYR10 is precisely regulated by drought and ABA signals at the transcriptional level.

[0131] Transcriptional alterations of drought-responsive genes rely on cis-acting regulatory elements located in their promoters. Elements such as drought and ABA-responsive elements (DRE), recognition sites for transcription factors MYB and MYC play key roles in regulating gene drought responses at the transcriptional level (Cutler et al., 2010; Singh and Laxmi, 2015). In this study, our analysis revealed a set of conserved elements in the TaPYR10 promoter that control gene transcription, including TATA and CAAT boxes, as well as elements that trigger gene responses to ABA and drought stress, namely DRE and recognition sites for MYB and MYC transcription factors. Further GUS histochemical staining and activity assays by transforming wheat leaves with a series of truncated promoter fragments driving the expression of the reporter gene GUS verified their functions in regulating gene responses to drought and ABA signals. Additional experiments using base mutation methods can help determine the mechanisms by which these cis-elements control gene transcription under drought stress.

[0132] (2) TaPYR10 and its downstream partners TaPP2C30, TaSnRK2.10, and TaNF-YC1 constitute the core ABA signaling pathway. An ABA signaling pathway functional module involved in mediating ABA and drought responses has been established in plant species (Asad et al., 2019). Under stress conditions, the accumulation of ABA and the formation of the PYR / PYL / RCAR-PP2C complex lead to the inactivation of PP2C, followed by the activation of SnRK2. Activated SnRK2 promotes the transcription of ABA-responsive genes by phosphorylating downstream substrates (Ali et al., 2020). However, in cereal crops, the downstream partners of ABA signaling members interacting with ABA receptors remain largely unknown. In this study, we performed protein-protein interaction analysis using a series of experiments, including yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and in vitro pull-down assays, to determine the components of the wheat ABA signaling pathway involving TaPYR10. Our results showed that TaPYR10 interacts with its downstream partners, namely TaPP2C30, TaSnRK2.10, and TaNF-YC1. These findings indicate the existence of a wheat ABA signaling pathway composed of these proteins (TaPYR10 / TaPP2C30 / TaSnRK2.10 / TaNF-YC1). Previous literature has reported that ABA receptors can function in the nucleus (Cutler et al., 2010), and most members of the PP2C family genes have also been found to be localized in the nucleus. The intracellular localization of ABA signaling partners contributes to the specificity of PP2C-RCAR pairing and its binding properties with downstream-acting SnRK2 (Tischer et al., 2017). In this study, our BiFC experiments revealed the interaction sites of TaPYR10 and its downstream partners in the nucleus. This is consistent with the localization reported in some previous studies, indicating that this may be the location where they perform their biological functions. To date, research on the different regions of protein-protein interactions between ABA receptors and their downstream partners remains limited. In this study, we determined the fragments involved in protein-protein interactions between the TaPYR10 protein and its downstream partners. Our results showed that the middle part of TaPYR10 (amino acids 71 to 140), the middle part of TaPP2C30 (amino acids 74 to 317), the middle part of TaSnRK2.10 (amino acids 91 to 260), and the N-terminus of TaNF-YC1 (amino acids 1 to 80) contribute to the protein-protein interactions between TaPYR10 and its downstream partners. Further characterization of the fine localization controlling the interactions in these fragments can help understand the molecular processes of protein-protein interactions in the ABA signaling pathway in common wheat.

[0133] (3) TaPYR10 and its downstream partners have a significant impact on plant drought response by regulating stress-related physiological indicators. The plant drought and ABA responses mediated by ABA receptor genes occur through the ABA-dependent signaling pathway (Park et al., 2009; González-Guzmán et al., 2014). Given their significant involvement in regulating stress defense physiological processes, including stomatal movement, osmolyte biosynthesis, cellular reactive oxygen species homeostasis, and root system architecture (RSA) (Pei et al., 2012; Li et al., 2014; Zhou and Luo, 2018), various ABA receptor genes have been identified as potential candidate genes for engineering plants with enhanced drought tolerance and improved water use efficiency (Mega et al., 2019; Yang et al., 2019). For example, the PYR / PYL / RCAR abscisic acid receptors in tomato are highly expressed in roots and have the ability to enhance plant drought tolerance (González-Guzmán et al., 2014). PePYL4 enhances the drought tolerance of poplar by regulating water use efficiency and reactive oxygen species scavenging (Li et al., 2022). These studies have deepened our understanding of the mechanism by which PYR1 / PYL / RCAR genes regulate plant drought adaptation. Similarly, studies on genes downstream of ABA receptors have also shown that they play a key role in mediating drought responses. The SnRK2 family member TaSnRK2.9 confers drought tolerance to transgenic tobacco plants (Feng et al., 2019). The PP2C family member MePP2C24 in cassava affects plant drought response through the ABA-dependent pathway (Zeng et al., 2024). In this study, we constructed transgenic lines of TaPYR10 and its downstream partners, namely TaPP2C30, TaSnRK2.10, and TaNF-YC1, including overexpression and knockdown lines, to characterize their functions in mediating drought responses. Our results verified the positive roles of TaPYR10, TaSnRK2.10, and TaNF-YC1, while demonstrating the negative function of TaPP2C30 in regulating plant growth under drought conditions. Our drought treatment of TaPYR10 transgenic lines under field conditions verified the positive role of this wheat PYR member in mediating plant adaptation to drought stress. These transgenic analysis results indicate that these genes are valuable for engineering drought-tolerant varieties of common wheat. The transgenic analysis of TaPYR10, TaPP2C30, TaSnRK2.10, and TaNF-YC1 in regulating drought responses in this study confirmed their roles in regulating these physiological processes.

[0134] To characterize the molecular processes of the TaPYR10 module in regulating osmotic stress-related physiological processes, we analyzed the expression patterns of each gene component to evaluate its impact on each trait, i.e., the slow anion channel 1 (SLAC1) family genes (TaSLAC1-1 to TaSLAC1-6) regulate stomatal movement by modulating stress-induced anion efflux (Linder and Raschke, 1992; Schroeder and Keller, 1992), proline accumulation-related genes (TaP5CS1, TaP5CS2, TaP5CR1, TaProDH1, TaP5CDH1) as limiting factors in proline biosynthesis (Bandurska et al., 2017), antioxidant enzyme (AE) members such as SOD and CAT family genes affect cellular reactive oxygen species homeostasis (Hong et al., 2024), and PIN-FORMED (PIN) family genes control cellular auxin transport and RSA behavior (Zhang et al., 2019; Pei et al., 2012; Li et al., 2014; Zhou and Luo, 2018). In TaNF-YC1 transgenic lines under drought stress, specific gene family members including TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 were differentially expressed. Compared with the wild type, after drought treatment, the transcripts of these genes were higher in the transgenic TaNF-YC1 overexpression lines and lower in the knockdown lines. These results indicate that these genes have potential roles in the drought response regulated by TaNF-YC1. We conducted yeast one-hybrid assays to determine the protein / DNA interactions between TaNF-YC1 and the promoter regions of these stress defense genes to understand whether these genes are regulated by wheat NF-Y transcription factors at the transcriptional level. Our results showed that TaNF-YC1 interacted with the promoters of these genes by specifically binding to different promoter regions containing ABRE or MYB, and ABRE and MYB are cis-regulatory elements responsive to drought and ABA signals, respectively. In addition, our transcriptional activation assays in tobacco (i.e., TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) verified that they are all regulated by TaNF-YC1. Moreover, transgenic analysis confirmed their biological roles in regulating stomatal closure (TaSLAC1-3), proline accumulation (TaP5CR1), antioxidant enzyme activities of SOD (TaSOD4) and CAT (TaCAT2), and root morphology (TaPIN6). In summary, the TaPYR10 module plays a key role in regulating plant drought responses by modulating a series of stress-responsive genes regulated by TaNF-YC1 at the transcriptional level.Altered expression of TaPYR10 and stress-responsive genes contributes to differential drought tolerance in wheat varietiesGenetic variation is considered a fundamental element for enhancing crop quantitative traits, including yield components and drought tolerance. Various methods have been adopted to broaden the genetic variation of crop plants, such as introducing existing varieties, developing segregating materials through local or international nurseries, hybridization, and mutagenesis breeding (Thudi et al., 2021). Additionally, using parental lines with diverse genetic backgrounds, including unrelated and complementary genetic resources with suitable drought adaptation and yield-increasing traits, can help create elite breeding populations (Thudi et al., 2021). In this study, we evaluated the expression levels of TaPYR10 and stress-responsive genes (i.e., TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) in a core-variety panel consisting of 45 wheat varieties with different plant drought responses. In a field experiment under water-saving irrigation management, at the mid-filling stage, significant differences in the expression levels of these genes were observed among varieties in the flag leaves. Moreover, a highly positive correlation was found between plant yield and the expression levels of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. Collectively, these findings indicate that the genes TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 play crucial roles in regulating plant yield production. Our findings suggest that the transcript abundance of the signaling module composed of TaPYR10 can serve as an effective indicator for evaluating the drought adaptation ability of wheat varieties grown under drought conditions. Based on the characterization of SNP behavior in the promoter of TaPYR10 in a wheat variety panel, we revealed base variations in the flanking sequence region of the MYB recognition site in the promoters of wheat varieties with different drought tolerances. Two haplotypes, including TaPYR10-Hap1 and TaPYR10-Hap2, were found to affect plant drought responses by influencing the transcript abundance of target genes. The haplotype TaPYR10-Hap1 confers better drought adaptation in wheat varieties, indicating that this haplotype may be a valuable target for molecular breeding of drought-tolerant varieties in common wheat. Based on our study, we established a working model of TaPYR10 and its downstream partners in mediating plant drought responses (). Figure 21)。In plant tissues, the TaPYR10 transcript is up-regulated under drought and ABA signal stimulation. After the induced TaPYR10 protein binds to ABA molecules, it interacts with the PP2C family member TaPP2C30 through a protein-protein interaction mechanism. The formed trimeric complex TaPYR10 / ABA / TaPP2C30 eliminates the inhibitory effect of TaPP2C30 on the common wheat SnRK2 family member TaSnRK2.10. Then TaSnRK2.10 interacts with the transcription factor member TaNF-YC1 of the common wheat NF-Y family, and TaNF-YC1 participates in regulating the biosynthesis of osmoregulatory substances, stomatal movement, and cellular reactive oxygen species homeostasis by transcriptionally activating the expression of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. The above-changed physiological processes contribute to plant adaptation to drought stress. Further identification and functional analysis of the molecular processes of the signaling pathway involved in TaPYR10 can provide new insights into the plant drought tolerance mechanism.

[0135] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions, and variations can be made to these examples without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

[0136] In the above embodiments, the descriptions of the respective embodiments have their own emphases. For parts not detailed or recorded in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0137] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the respective embodiments of the present invention, and should all be included in the protection scope of the present invention.

Claims

1. A functional module for regulating plant drought response, characterized in that: Including TaPYR10 and its downstream signaling pathway members.

2. The functional module according to claim 1, characterized in that: The downstream signaling pathway members include TaPP2C30, TaSnRK2.10 and TaNF-YC1.

3. A method for cultivating drought-resistant transgenic plants, characterized in that: Gene editing of the TaPYR10 gene and its downstream signaling pathway members produced transgenic plants with drought-resistant characteristics.

4. The method for cultivating drought-resistant transgenic plants according to claim 3, characterized in that: Specifically, the TaPYR10, TaSnRK2.10 and TaNF-YC1 genes are overexpressed, and the TaPP2C30 gene is inhibited, reduced, silenced or knocked out.

5. A kit for regulating plant drought response, characterized in that: The kit contains molecular biological elements capable of regulating the expression of specific genes; the specific genes are genes related to the drought response regulating ability of plants.

6. The kit according to claim 5, characterized in that: The molecular biological element is a combination of elements for overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibiting, reducing, silencing or knocking out TaPP2C30 gene.

7. A method for enhancing plant drought stress tolerance, characterized in that: Overexpression of TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibition or reduction or silencing or knockout of TaPP2C30 gene can enhance the drought stress tolerance of plants.

8. A method for increasing plant yield, characterized in that: Overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out the TaPP2C30 gene can enhance the drought stress tolerance of the corresponding plants, thereby increasing the yield of the corresponding plants.

9. The functional module according to any one of claims 1-2, the method according to any one of claims 3-4, the kit according to any one of claims 5-6, the method according to claim 7, or the method according to claim 8, characterized in that: The plant is wheat.

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