Functional module for regulating drought response of plants and use thereof

Abstract

The application discloses a functional module for regulating drought response of plants and application thereof, and the functional module comprises TaPYR10 and downstream signal path members thereof. The application discloses the role of TaPYR10 and downstream partners (TaPP2C30, TaSnRK2.10 and TaNF-YC1) in drought signal conduction, and provides a new molecular basis for drought tolerance of wheat. The functional analysis of the genes shows that the genes play a key role in the response of plants to drought stress, and can improve the stress resistance of plants by regulating relevant physiological processes (such as stomatal movement, synthesis of osmoregulation substances and cell active oxygen homeostasis).

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly to a functional module for regulating plant drought response and application thereof. BACKGROUND

[0002] Drought stress, caused by reduced precipitation and limited water supply, is one of the most destructive abiotic stresses that adversely affects plant growth, development, and crop yield formation. In recent years, global climate change is expected to exacerbate the degree of water stress on 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 current wheat planting areas. Therefore, the use of 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 significant effects on physiological, biochemical, and molecular processes related to water relations at the cellular, tissue, and organ levels. For major crops, drought can cause significant decreases in relative water content, chlorophyll content, and plant productivity. Therefore, plants have evolved various strategies to mitigate the negative effects of drought by accumulating osmotic adjustment substances (such as proline, soluble sugars, betaine, organic acids, free amino acids, and other organic compounds) to enhance their osmotic adjustment capacity to maintain adequate water supply. Plants can also activate antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) to improve reactive oxygen species scavenging capacity and maintain reactive oxygen species homeostasis. In addition, plant hormones such as auxin (IAA), gibberellin (GA), zeatin riboside (ZR), abscisic acid (ABA), and jasmonic acid (JA) synergistically mediate plant drought response.

[0004] Abscisic acid (ABA) is a major stress-responsive hormone produced by plants in response to drought, which influences multiple physiological processes throughout the plant life cycle, including seed dormancy and germination, root growth, stomatal guard cell swelling, senescence, and fruit ripening. ABA-mediated stress responses are achieved through a specific ABA signaling pathway, in which plant cells perceive ABA signals and convert them into various physiological and biochemical responses through a signal transduction process. When plants are exposed to drought conditions, members of the ABA receptor family (i.e., PYR / PYL / RCAR) play a role in perceiving drought signals in the environment and triggering ABA signal transduction through a protein phosphorylation mechanism. It has been confirmed that PYR / PYL / RCAR members, 2C-type protein phosphatase (PP2C) proteins, and members of the SNF1 -related protein kinase 2 (SnRK2) family are 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 adequate 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 physiological, biochemical, and molecular processes involved in plant drought responses. In addition, under drought conditions, the ABA concentration in tissues increases, then binds to PYR / PYL / RCAR proteins, allowing the ABA receptor to interact with PP2C proteins, resulting in the release of SnRK2 from the inhibition of PP2C, thereby activating downstream targets such as ABA-responsive element binding factors (ABFs), ABA-responsive element binding proteins (AREBs), and members of the NAC, MYC, and MYB transcription factor families. Thus, ABA receptor-mediated signaling pathways are key regulators of plant drought responses.

[0005] Recent studies have identified and characterized NF-Y family members as well as PP2C and SnRK2 family members in the model plant Arabidopsis thaliana. Functional characterization of NF-Y genes indicates that they play a key role as mediators of plant drought stress response as they regulate biological processes associated with drought stress response. Among the NF-Y genes in maize, ZmNF-YA1 and ZmNF-YB16 have been shown to confer better drought adaptation to plants by enhancing the transcription of osmotic stress responsive genes and improving water use efficiency of plants under drought stress. The ABA receptor family member PYR / PYL / RCAR is downstream of NF-Y transcription factors, where 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 active oxygen (such as h2O2) levels and improve plant productivity under drought conditions. In Arabidopsis thaliana, ten SnRK2 family members have been identified, and expression analysis shows that all SnRK2 members in Arabidopsis are activated by osmotic stress, except for SnRK2.9, and functional characterization confirms that they are involved in transducing osmotic stress signals through an ABA-dependent pathway. In addition, A-type PP2C proteins in Arabidopsis play a crucial role as negative regulators in the ABA signaling pathway. Therefore, different members of the ABA receptor family and downstream partners form a signaling pathway that mainly regulates stress defense biological processes through an ABA-dependent pathway, thereby contributing to plant drought tolerance.

[0006] Wheat is an important crop widely planted worldwide. With the intensification of global warming, soil drought is becoming increasingly serious, which has a negative impact on wheat yield. 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, the functional characterization of ABA receptor family members and their downstream partners in the PP2C and SnRK2 families, as well as transcription factor members involved in plant drought defense, is still limited in common wheat. SUMMARY

[0007] The technical problem to be solved by the present application 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 application are as follows.

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

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

[0011] The application discloses a method for cultivating drought-resistant transgenic plants, gene editing TaPYR10 and downstream signal pathway members thereof, and cultivating transgenic plants with drought-resistant characteristics.

[0012] Further preferably, the application specifically overexpresses TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibits or reduces or silences or knocks out TaPP2C30 genes.

[0013] The application discloses a kit for regulating drought response capability of plants, wherein the kit comprises a molecular biology element capable of regulating expression amount of specific genes; and the specific genes are genes related to drought response capability of plants.

[0014] Further preferably, the application specifically overexpresses TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibits or reduces or silences or knocks out TaPP2C30 genes.

[0015] The application discloses a method for enhancing drought stress tolerance of plants, overexpresses TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibits or reduces or silences or knocks out TaPP2C30 genes, so as to enhance drought stress tolerance of plants.

[0016] The application discloses a method for improving yield of plants, overexpresses TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibits or reduces or silences or knocks out TaPP2C30 genes, so as to enhance drought stress tolerance of corresponding plants, and thus improve yield of the corresponding plants.

[0017] According to the functional module, the method, the kit, the method or the method described above, the plants are wheat.

[0018] The application discloses a use of a functional module comprising TaPYR10 and downstream signal pathway members thereof, evaluates drought response capability of plants by analyzing expression level of TaPYR10 and downstream signal modules, and screens, preliminarily screens, identifies or assists in identifying wheat germplasm resources with strong drought resistance, specifically, screens, preliminarily screens, identifies or assists in identifying wheat germplasm overexpressing TaPYR10, TaSnRK2.10 and TaNF-YC1 genes, and inhibiting or reducing or silencing or knocking out TaPP2C30 genes.

[0019] The application discloses a method for enhancing drought stress tolerance of wheat, introduces multiple copies of cis-elements DRE and MYB and MYC TF protein binding sites into a wheat genome by gene editing technology, constructs a system capable of overexpressing TaPYR10 under drought and ABA signal stimulation, and enhances drought stress tolerance of wheat.

[0020] A SNP locus associated with wheat proline content, biomass, and yield, characterized in that: the SNP locus is located on wheat chromosome 1; the physical distance of the SNP locus in wheat genome version number Triticum aestivumiwgsc_refseqv1.0 is Chr1A_346681497; the four SNP loci of ORF in TaPYR10 correspond to bases 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, and A / A in sequence, the corresponding genotype is Hap1; when the loci are homozygous for T / T, A / A, C / C, and G / G, the corresponding genotype is Hap2; the five SNP loci in the promoter region correspond to SEQ ID NO. In the sequence shown in NO.9, the 1680th, 1684th, 1685th, 1737th, and 1902nd bases from the end of the sequence, when these sites are homozygous for T / T, A / A, C / C, A / A, and G / G respectively, correspond to the genotype Hap1. When the sites are homozygous for C / C, G / G, T / T, G / G, and T / T respectively, the corresponding genotype is Hap2. The proline content, biomass, and yield are as follows: wheat homozygous for genotype Hap1 is greater than or candidate greater than wheat homozygous for genotype Hap2.

[0021] A primer combination for detecting single nucleotide polymorphism of SNP sites in a wheat genome, wherein the SNP sites in the wheat genome version number Triticum aestivum iwgsc_refseqv1.0 have a physical distance of Chr1A_346681497, four SNP sites of the 0RF in the TaPYR10 correspond to bases at positions 211, 224, 374 and 410 in the sequence shown in SEQ ID NO. 1, and when the sites are G / G, T / T, G / G and A / A in order, the corresponding genotype is Hap1, and when the sites are T / T, A / A, C / C and G / G in order, the corresponding genotype is Hap2; five SNP sites in the promoter region correspond to bases at positions 1680, 1684, 1685, 1737 and 1902 from the end of the sequence shown in SEQ ID NO. 9, and when the sites are T / T, A / A, C / C, A / A and G / G in order, the corresponding genotype is Hap1, and when the sites are C / C, G / G, T / T, G / G and T / T in order, the corresponding genotype is Hap2; the proline content, biomass and yield size of the genotype Hap1 homozygous wheat are greater than or greater than the genotype Hap2 homozygous wheat; the primer combination is a primer pair consisting of SEQ ID NO. 244 to SEQ ID NO. 279 in the sequence listing; and the primer combination is used for detecting SNP sites.

[0022] According to the SNP site, a Kasp molecular marker related to drought resistance of wheat is developed.

[0023] A method for breeding drought-tolerant and high-yield wheat varieties, comprising the following steps: in the early breeding stage, detecting the transcript abundance of TaPYR10 gene and stress defense genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 in a wheat hybrid offspring population; according to the detection results, preferentially selecting individuals with transcript abundance of TaPYR10 gene and the stress defense genes higher than a pre-set threshold value from the hybrid offspring population, wherein the pre-set threshold value is determined by collecting a large number of wheat varieties with different drought tolerance and yield performance, testing the transcript abundance of each gene in the upper leaves of the middle grain-filling stage under field drought conditions and the yield data at maturity, and statistically analyzing; breeding the selected individuals to obtain wheat varieties or lines with high drought tolerance and high yield characteristics, and the breeding efficiency is greatly improved and the breeding period is shortened by the method.

[0024] The beneficial effects produced by the above technical scheme are that in the present research, we focus on characterizing the ABA receptor gene TaPYR10 in wheat to identify its downstream partners and functionally characterize the ABA core signal module in mediating plant drought response. The research results of the present application provide new insights into the mechanism of the ABA signaling pathway in plant drought response, and may promote the development of drought-tolerant varieties of common wheat through molecular breeding;

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

[0026] The present application provides a new insight into the gene regulation mechanism of plants under environmental stress by analyzing the cis-regulatory elements in the TaPYR10 promoter, and provides a new target for genetic engineering and breeding to improve breeding efficiency;

[0027] The present application verifies the function of TaPYR10 and its downstream partners in drought response by constructing transgenic wheat lines, and provides genetic resources for subsequent crop improvement, especially in improving the drought tolerance of wheat, which has important application prospects. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0030] Figure 3Figure 10 is a schematic diagram showing the expression pattern of TaPYR10 under drought and ABA treatment, wherein Figure A-B are a schematic diagram showing the expression pattern of TaPYR10 in roots and leaves under drought treatment (Figure A) and under ABA treatment (Figure B); in Figure A, 0, 1%, 5%, 10% and 15% represent the concentration of PEG (w / v), 1 h, 3 h, 9 h and 27 h represent the time after drought treatment (10% PEG, w / v), R1 h, R3 h, R9 h and R27 h represent the time of recovery treatment after 27 h drought challenge, 0 h represents the time point before drought treatment; in Figure B, 0, 0.5 μmol, 1 μmol, 1.5 μmol and 2 μmol represent the concentration of exogenous ABA, 1 h, 3 h, 9 h and 27 h represent the time after ABA treatment (1.5 μmol ABA); Figure C is a schematic diagram of a binary expression cassette integrated with TaPYR10 promoter; Figure D is a schematic diagram showing the cis-regulatory elements related to osmotic stress, i.e. MYB (CAACAG, CAACCA and CCGTTG), DRE (GCCGAC) and MYC (TCTCTTA) located in the TaPYR10 promoter; Figure E-F are a schematic diagram showing 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 (454 bp, 745 bp, 1378 bp, 1666 bp and full length 1764 bp) of the TaPYR10 promoter, in Figure F, the data are represented as mean ± standard deviation (n = 3), the statistical significance between transgenic lines under the same growth condition was tested using Student's t-test (*p < 0.05);

[0031] Figure 4Figure 6 shows the results of protein interaction experiments between TaPYR10 and its downstream partners; Figure 6A is a schematic diagram showing the predicted interaction between TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins; Figures 6B-6D are schematic diagrams showing the results of yeast two-hybrid experiments indicating the interaction between TaPYR10 and TaPP2C30 (Figure 6B), TaPP2C30 and TaSnRK2.10 (Figure 6C) and TaSnRK2.10 and TaNF-YC1 (Figure 6D); Figure 6E is a schematic diagram showing the results of BiFC experiments verifying the interaction between TaPYR10 and its downstream partners; Figures 6F-6H are schematic diagrams showing the results of co-immunoprecipitation experiments indicating the interaction between TaPYR10 and TaPP2C30 (Figure 6F), TaPP2C30 and TaSnRK2.10 (Figure 6G) and TaSnRK2.10 and TaNF-YC1 (Figure 6H); Figures 6I-6K are schematic diagrams showing the results of in vitro pull-down experiments indicating the protein interaction between TaPYR10 and TaPP2C30 (Figure 6I), TaPP2C30 and TaSnRK2.10 (Figure 6J) and TaSnRK2.10 and TaNF-YC1 (Figure 6K);

[0032] Figure 5Figure 1 is a schematic diagram of experimental results for TaPYR10 and its downstream partner-interacting protein fragments; wherein Figure A is a schematic diagram of various fragments of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins, which were used for yeast two-hybrid and BiFC experiments; Figure B is a schematic diagram of yeast two-hybrid experimental results, indicating the contribution of different fragments of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins to protein interaction; Figure C is a schematic diagram of BiFC experimental results, verifying the fragments involved in the interaction between TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 proteins; in Figure B and Figure 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); Figure D-F are schematic diagrams of co-immunoprecipitation experimental results, indicating different fragments involved in protein interaction between TaPYR10 (TaPYR10I1) and TaPP2C30 (TaPP2C30I1) (Figure D), TaPP2C30 (TaPP2C30I1) and TaSnRK2.10 (TaSnRK2.10I1) (Figure E), and TaSnRK2.10 (TaSnRK2.10I1) and TaNF-YC1 (TaNF-YC1N1) (Figure F); Figure G-I are schematic diagrams of in vitro pull-down experimental results, verifying the fragments involved in the interaction 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 Figure 2 is a schematic diagram of experimental results for evaluating the interaction between TaPYR10 and PP2C, SnRK2 and NF-YC family proteins by yeast two-hybrid method, wherein 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 7Figure 1. Schematic representation of the expression levels of the target genes TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 in transgenic wheat lines. Figures A-D show the expression levels of the target genes in the following transgenic lines: A, TaPYR10 transgenic line; B, TaPP2C30 transgenic line; C, TaSnRK2.10 transgenic line; D, TaNF-YC1 transgenic line. The expression values were normalized to Tatubulin and Taactin, whose expression levels were set to 1. The data are means ± 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 Figure 2. Schematic representation of the phenotype and growth characteristics of the transgenic lines of TaPYR10 and its downstream partner genes under drought treatment. Figures A-D show the phenotype results of the plants at the mid-grain filling stage under drought treatment. Figures E-H show the plant biomass results of the plants at the mid-grain filling stage when grown in the field. Figures I-L show the leaf area results of the plants at the mid-grain filling stage when grown in the field. Figures M-P show the grain weight results of the plants grown in the field. Figures Q-T show the yield results of the plants grown in the field. In Figures E-T, the soil water content under normal conditions was 70-75% of the field capacity. In Figures A-T, the drought treatment was performed with a soil moisture of 55-60% of the field capacity. The data are means ± standard deviation (n = 3). The statistical significance between the transgenic lines and the WT under the same growth conditions was tested using Student's t-test (*p < 0.05).

[0036] Figure 9 Figure 3. Schematic representation of the plant phenotype of the transgenic lines (overexpression or knockdown) of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 grown in the field under normal conditions.

[0037] Figure 10Figure 9 is a schematic diagram of the phenotype and plant biomass of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under exogenous ABA treatment; wherein Figure A is a schematic diagram of the phenotype results of TaPYR10 transgenic lines; Figure B is a schematic diagram of the phenotype results of TaPP2C30 transgenic lines; Figure C is a schematic diagram of the phenotype results of TaSnRK2.10 transgenic lines; Figure D is a schematic diagram of the phenotype results of TaNF-YC1 transgenic lines; Figure E is a schematic diagram of the plant biomass results of TaPYR10 transgenic lines; Figure F is a schematic diagram of the plant biomass results of TaPP2C30 transgenic lines; Figure G is a schematic diagram of the plant biomass results of TaSnRK2.10 transgenic lines; Figure H is a 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, data are presented as mean ± standard deviation (n = 3); statistical significance between transgenic lines and WT was tested using Student's t-test (*p < 0.05 and **p < 0.01);

[0038] Figure 11 Figure 10 is a schematic diagram of the osmotic stress-related physiological characteristics of TaPYR10 and its downstream partners transgenic lines, wherein Figure A is a schematic diagram of stomata characteristics results under drought stress; Figures B-E are schematic diagrams of stomata closure rate results during 2 hours of drought treatment; Figures F-I are schematic diagrams of leaf water loss results during 2 hours of water loss; Figures J-M are schematic diagrams of proline content results; Figures N-Q are schematic diagrams of soluble sugar content results; in Figures B-Q, data are presented as mean ± standard deviation (n = 3); statistical significance between transgenic lines and WT under similar conditions was tested using Student's t-test (*p < 0.05);

[0039] Figure 12 Figure 11 is a schematic diagram of the photosynthetic parameters results of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment, wherein Figures A-D are schematic diagrams of net photosynthetic rate (Pn) results; Figures E-H are schematic diagrams of actual quantum efficiency of photosystem II (ΦPSII) results; Figures I-L are schematic diagrams of stomatal conductance (Gs) results; Figures M-P are schematic diagrams of non-photochemical quenching coefficient (NPQ); data are presented as mean ± standard deviation (n = 3); different lower case letters indicate significant differences between transgenic lines at the same growth stage (Tukey's test, P < 0.05);

[0040] Figure 13Figure 9 is a schematic diagram of ROS-related indicators and root phenotypes of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment; wherein Figures A-D are schematic diagrams of results of superoxide anion content assessed by nitro blue tetrazolium (NBT) staining; Figures E-H are schematic diagrams of results of H2O2 content assessed by 3,3-diaminobenzidine (DAB) staining; Figures I-L are schematic diagrams of results of superoxide dismutase (SOD) activity; Figures M-P are schematic diagrams of results of catalase (CAT) activity; Figures Q-T are schematic diagrams of results of crude enzyme (POD) activity; Figures U-X are schematic diagrams of results of root phenotypes; in Figures I-T, data are represented as mean ± standard deviation (n=3) and statistical significance between WT and transgenic lines was tested using Student's t-test (*p<0.05);

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

[0042] Figure 15 Figure 11 is a schematic diagram of results 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 results of MDA content; Figures E-H are schematic diagrams of results of root biomass; Figures I-L are schematic diagrams of results of root volume; data are represented as mean ± standard deviation (n=3) and different lower case letters indicate significant differences between transgenic lines under the same growth condition (Tukey's test, P<0.05);

[0043] Figure 16Figure A-E are the expression profiles of 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 the schematic diagram of cis-acting elements located 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 the schematic diagram of the results of yeast one-hybrid experiment, the results show 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 the triple dropout medium (SD / -Leu-Trp-His) plates containing 45mM 3-AT; Figure H-L are the schematic diagrams of luciferase signals detected after co-transforming the effectors CaMV35SPro::TaNF-YC1 and the reporter genes TaSLAC1-3Pro::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; Figure M-Q are the results of LUC activity in leaves co-transformed with the combinations of effectors and reporter genes mentioned in Figure H-L; the data in Figure M-Q are expressed as mean ± standard deviation (n = 3), and Student's t-test was used to test the statistical significance between LUC activities caused by effectors / reporter genes (*p < 0.05);

[0044] Figure 17Figure 1. Physiological parameters and root morphological characteristics of the gene knockdown lines under drought treatment. Figure A-C are the results of stomatal morphology (Figure A), stomatal aperture (Figure B), and transpiration rate (Figure C) of TaSLAC1-3 knockdown lines within 1 hour of drought treatment. AntiSLAC1-3A and AntiSLAC1-3B are TaSLAC1-3 knockdown lines. Figure D-F are the results of phenotype (Figure D), plant biomass (Figure E), and proline content (Figure F) of TaP5CR1 knockdown lines at the seedling stage. AntiP5CR1-1 and AntiP5CR1-2 are TaP5CR1 knockdown lines. Figure G-I are the results of phenotype (Figure G), superoxide dismutase (SOD) activity (Figure H), and histochemical staining to show accumulated superoxide anion (Figure I) of TaSOD4 knockdown lines at the seedling stage. AntiSOD4-2 and AntiSOD4-3 are TaSOD4 knockdown lines. Figure J-L are the results of phenotype (Figure J), catalase (CAT) activity (Figure K), and histochemical staining to show accumulated H2O2 (Figure L) of TaCAT2 knockdown lines at the seedling stage. AntiCAT2-2 and AntiCAT2-3 are TaCAT2 knockdown lines. Figure M-O are the results of root morphology (Figure M), root biomass (Figure N), and root volume (Figure O) of TaPIN6 knockdown lines grown under field conditions. AntiPIN6-1 and AntiPIN6-2 are TaPIN6 knockdown lines. Data are means ± standard deviation (n = 3), and statistical significance of transgenic lines and wild type under the same growth conditions was tested using Student's t-test (*p < 0.05).

[0045] Figure 18 Figure 2. Superoxide anion and H2O2 contents in TaSOD4 and TaCAT2 knockdown lines under drought treatment.

[0046] Figure 19Figure A shows the expression level results of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 in the tested wheat varieties; Figure B shows the yield results in the tested wheat varieties; Figures C-H are the regression analysis results of yield and TaPYR10 transcripts (Figure C), yield and TaSLAC1-3 transcripts (Figure D), yield and TaP5CR1 transcripts (Figure E), yield and TaSOD4 transcripts (Figure F), yield and TaCAT2 transcripts (Figure G), and yield and TaPIN6 transcripts (Figure H), respectively; in Figures A-B, the left side shows 45 tested wheat varieties and is simplified according to the registered name;

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

[0048] Figure 21 Figure is a working model of TaPYR10 and its downstream partners in mediating plant drought response. DETAILED DESCRIPTION

[0049] The following examples illustrate the present application in detail. The various raw materials and equipment used in the present application are all conventional commercially available products, which can be directly obtained by market purchase. The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0050] It should be understood that when used in the specification and the appended claims, the term "comprises" indicates the presence of the described features, integers, steps, operations, elements, and / or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0051] It should also be understood that, in the description of the application and the appended claims, the terms "and / or" are used to mean one or more of the items in the list joined by "and / or" and that it is possible for the items in the list to be combined in any of the ways that the item is associated with "and / or". It should also be understood that, in the description of the application and the appended claims, the term "comprises" is used to mean that the item that follows the term "comprises" is not the only item that can be included in the item that follows the term "comprises".

[0052] Reference throughout this specification to "one embodiment" or "some embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the application. Thus, the appearances of the phrases "in one embodiment" or "in some embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment, but can refer to one or more but not all embodiments. Furthermore, the terms "comprises an," "comprises," "comprising," "includes," "including," "has," "having" or variants thereof are intended to mean "including but not limited to," unless expressly specified otherwise.

[0053] In addition, the description in the specification and the appended claims of the application use the term "first", "second", "third", etc. only to distinguish descriptions, and cannot be understood as indicating or implying relative importance.

[0054] The technical solutions of the present application will be described clearly and completely below in combination with specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0055] Example 1, Materials and Methods

[0056] (1) Plant materials and growth conditions: The wheat (Shimai 22) seedlings grown in hydroponics in a growth chamber were used to analyze the expression of TaPYR10 under drought and ABA treatment. The transgenic lines 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 TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 regulated by TaNF-YC1 were grown 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 response.

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

[0058] The above transgenic lines and the core wheat variety population including 45 varieties were grown for the evaluation of growth traits and gene expression levels in field experiments, which were conducted at the Experimental Station of the Institute of Crops and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences, Shijiazhuang City, Hebei Province, China during the 2022-2023 and 2023-2024 planting seasons (North Latitude 37°56'24.62", East Longitude 114°42'46.96").

[0059] Table 1 Information of the core wheat variety population of 45 varieties participating in the field test

[0060]

[0061]

[0062]

[0063] (2) Molecular characterization analysis

[0064] The molecular properties of the wheat ABA receptor (PYR) family member TaPYR10 (GenBank Accession No. TraesCS1A02G191700) were characterized as follows: The open reading frame (ORF) of TaPYR10 and its 2 kb long promoter region (nucleotide sequence as shown in SEQ ID NO. 9) were retrieved from the Ensembl Plants website (www.http: / / plants.ensembl.org / index.html). Conserved domains of the TaPYR10 protein similar to other PYR counterparts were defined. The phylogenetic relationship between TaPYR10 and its homologous genes in different plant species was analyzed by BLASTn search analysis on the GenBank database of the National Center for Biotechnology Information (NCBI, https: / / www.blast.ncbi.nlm.nih.gov / Blast.cgi) and their association was established using the MegAlign algorithm in the DNAStar software (https: / / www.dnastar.com). ClustalW alignment analysis of the TaPYR10 protein and its plant counterparts was performed using the 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 associated 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 epidermal cells of N. benthamiana. For this, the ORF of TaPYR10 was amplified using gene-specific primers based on reverse transcription polymerase chain reaction (RT-PCR) and integrated in-frame with a reporter gene (GFP) under the control of the CaMV35S promoter into the binary vector pCAMBIA3300. The resulting expression cassette TaPYR10-GFP was then transformed into Agrobacterium (strain EHA105) using the conventional heat shock method. Positive transformants were used for the transient transformation of epidermal cells of N. benthamiana using the Agrobacterium-mediated transformation method (Li et al., 2012). After 48 hours of transformation, the GFP signal in cells containing the TaPYR10-GFP fusion construct and cells with the empty vector integrated were observed using a fluorescence microscope.

[0067] Table 2 Gene-specific primers

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] (4) Expression analysis of TaPYR10

[0074] Three-leaf stage wheat (cv. Shimai 22) seedlings grown 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 included 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 hour before treatment and 1, 3, 9, and 27 hours 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-mentioned signal conditions. TaPYR10 transcripts in roots and leaves were detected based on quantitative reverse transcription polymerase chain reaction (qRT-PCR) using gene-specific primers (Table 2). Two constitutive genes, microtubulin (Ta tubulin) and actin (Ta actin) in wheat were used as internal controls to normalize the target transcripts.

[0075] (5) 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 located in TaPYR10 promoter from Plant Cis-acting Regulatory Element Database (PlantCARE, http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) were determined. For this, the full-length promoter (1764 bp) and a set of promoter regions of TaPYR10, i.e., 454 bp, 745 bp, 1378 bp, and 1666 bp, containing various cis-acting regulatory elements affecting gene transcription and osmotic stress response (i.e., DRE and MYB, MYC recognition sites) 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 reporter gene (beta-glucuronidase, GUS)) were obtained using Agrobacterium-mediated transformation method (Kumar et al., 2019). GUS histochemical staining and activity assessment of representative leaves of T2 transgenic lines were performed after 6 hours of drought treatment (10% PEG) (Houde et al., 2020).

[0077] Table 3. Table of drought-related cis-acting elements in TaPYR10 promoter and stress response-related gene information

[0078]

[0079]

[0080]

[0081] (6) Expression pattern analysis of TaPYR10 downstream partners

[0082] TaSLAC1 family members regulating stomatal movement, family members involved in proline biosynthesis, SOD family affecting superoxide dismutase activity, CAT family affecting catalase activity, and PIN-FORMED (PIN) family genes controlling auxin transport and root architecture behavior were analyzed for expression in TaNF-YC1 transgenic lines under drought stress. The SLAC1 genes analyzed included TaSLAC1-1 through TaSLAC1-6, the proline biosynthesis genes included TaP5CS1, TaP5CS2, TaP5CR1, TaProDH1, and TaP5CDH1, the SOD genes included TaSOD1 through TaSOD6, the CAT genes included TaCAT1 through TaCAT6, and the PIN genes included TaPIN1 through TaPIN6. Root tissue from three-leaf stage seedlings grown in modified MS solution (10% PEG) was evaluated for target expression levels after 27 hours of treatment. Similar to the qRT-PCR procedure performed for TaPYR10 under drought stress, transcripts of the above genes were detected using gene-specific primers (Table 2). The expression patterns of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, TaPIN6, and TaPYR10 were evaluated in 45 test wheat varieties (Table 1) at the grain-filling mid-stage under field drought conditions (soil water potential at the plow layer was -1.05 mPa). 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 by conventional deficit irrigation management (irrigation before sowing and at the jointing stage). Representative flag leaves of the varieties were collected using gene-specific primers (Table 2) and target transcripts were evaluated based on qRT-PCR.

[0083] (7) Yeast two-hybrid experiments

[0084] TaPYR10 was determined to be involved in the ABA signaling module using yeast two-hybrid experiments, i.e. PP2C proteins, SnRK2 kinases and NF-Y transcription factors. The PP2C family members used in the experiments include TaPP2C3, TaPP2C5, TaPP2C7, TaPP2C10 and TaPP2C30; the SnRK2 family members include TaSnRK2.5 to TaSnRK2.10; and the NF-Y family members include TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC5 and TaNF-YC8 (Table 2). For this purpose, the ORF of TaPYR10 was amplified using 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 the prey (each TaPP2C protein) was cultured on a selection solid medium supplemented with exogenous ABA (1 μmol) to initiate protein-protein interaction. Using a similar strategy, the ORFs of the PP2C members interacting 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. Likewise, the SnRK2 members interacting with the above-mentioned 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 the prey were cultured on a selection medium SD / -Leu / -Trp lacking specific amino acids at 30°C for 3 days. Fragments of TaPYR10 [i.e. TaPYR10-N1 (amino acids 1-70), TaPYR10-I1 (amino acids 71-140) and TaPYR10-C1 (amino acids 141-211)], TaPP2C30 [TaPP2C30-N1 (amino acids 1-73), TaPP2C30-I1 (amino acids 74-317) and TaPP2C30-C1 (amino acids 318-406)], TaSnRK2.10 [TaSnRK2.10-N1 (amino acids 1-90), TaSnRK2.10-I1 (amino acids 91-260) and TaSnRK2.10-C1 (amino acids 261-394)] and TaNF-YC1 [TaNF-YC1-N1 (amino acids 1-80) and TaNF-YC1-C1 (amino acids 81-255)] were amplified by RT-PCR using gene-specific primers (Table 2). They were then used to construct the bait or the prey, and the yeast two-hybrid experiments similar to the above were performed.

[0085] (8) BiFC experiment

[0086] BiFC experiments were performed to verify the in vivo protein interactions between TaPYR10 and its downstream partners. For this, the ORFs of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 were amplified using RT-PCR and gene-specific primers (Table 2) and integrated in frame with a fragment 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) according to the previously described method (Shen et al., 2011). The expression cassettes, i.e. 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 combined for genetic co- transformation of Agrobacterium (strain EHA105) and further transiently transformed into young leaves of N. benthamiana (Kumar et al., 2019). After infiltration for 2 days, the YFP signal in the transgenic leaves was detected using a fluorescence microscope (Olympus FV10-ASW, Japan) according to the manufacturer’s instructions. While detecting the YFP signal, the nuclear target was identified using the nuclear marker DAPI (Solarbio, Beijing, China) according to the previously described method (Sun et al., 2013). Various fragments of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 were amplified as in the above yeast two-hybrid experiment and used for BiFC experiments using gene-specific primers (Table 2). The YFP signal from the transformed young leaves of N. benthamiana was detected in a similar manner as above.

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

[0088] The ORFs of TaPYR10 and its downstream partners TaPP2C30, TaSnRK2.10 and TaNF-YC1 and 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). They were then used to construct expression cassettes, i.e. 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 Agrobacterium strain EHA105. The positive transformants were used for transient transformation of N. benthamiana leaves (Zhao et al., 2022). Briefly, about 0.3 g of leaves were collected after infiltration for 24 h and ground into powder using 1 mL of buffer A (50 mM hEPES-NaO h ph 7.5, 150 mM NaCl, 5% glycerol, 0.5% Triton X-100, 0.1% (v / v) 3-mercapto-1,2-propanediol, one tablet of Complete Mini protease inhibitor cocktail mixture (Shangon, Shanghai, China) per 10 mL). The resulting solution was centrifuged at 1000 g at 4 °C twice for 10 min to obtain the protein fraction. They were further mixed with an equal volume of 2x 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 before storage 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 with buffer A for 4-6 times, the proteins were eluted with 1x SDS-PAGE sample buffer (50% 2x SDS-PAGE sample buffer, 50% 1x PBS). Immunoprecipitation experiments were performed using 12.5% SDS-PAGE gels (e-PAGEL) (ATTO, Tokyo, Japan).

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

[0090] In vitro pull-down experiments were performed to validate the protein interactions shown in the above Co-IP experiments. The ORFs of TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 and cDNA sequences encoding different fragments of the above signaling 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 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 E. coli BL21 (DE3) strain for protein expression. The positive transformants were collected at 4 °C, 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 ice bath. The resulting supernatants were separated and subjected to SDS-PAGE for detecting protein components. Equal amounts of TaPYR10-GST and its downstream proteins were incubated in 500 μl GST pull-down buffer at 4 °C for 6 h, then 100 μl GST beads (GE healthcare, New Jersey, USA) were added to the mixture and incubated at 4 °C for 2 h. The pulled-down proteins were detected using anti-MBP antibody (New England Biolabs, Los Angeles, USA) and anti-GST antibody (Abeam, Cambridge, UK) according to the manufacturer's instructions.

[0091] (11) Construction of transgenic wheat lines

[0092] 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 were constructed to study their roles in mediating drought responses. Briefly, the ORFs of the above genes were amplified in forward or reverse direction by RT-PCR using gene-specific primers (Table 2). The amplification products were integrated into the Ncol / BastEII sites of the binary vector pCAMBIA3301 under the control of the CaMV35S promoter, respectively. The resulting expression cassettes were used to genetically transform Agrobacterium (strain EHA105), which was further used to transform common wheat (cv. Shimeizi 22) (Kumar et al., 2019). The target transcripts in transgenic lines were evaluated by qRT-PCR using gene-specific primers (Table 2). Representative lines of overexpression and knockdown of each gene, as well as wild type (WT), were selected in the T3 generation, cultured under normal conditions, and subjected to drought or ABA treatment.

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

[0094] Drought and ABA treatment 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, transgenic lines and wild type (WT) were grown under growth chamber or field conditions. For growth chamber culture, 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 70-75% relative soil humidity. 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 limiting water supply to maintain 55-60% relative soil humidity (detected using a soil water potential meter TRS-IIN, Zhejiang, China). For field planting, transgenic and WT plants were conventionally planted in field plots (1 m long and 0.3 m wide for each) with three replicates. Two irrigations (before sowing and at the jointing stage) were performed, which was considered as the conventional deficit irrigation management for local winter wheat (soil water potential at the plough layer was -1.05 to -1.22 mPa at the grain filling stage). ABA treatment was established by externally spraying 1 pmol ABA on seedlings or plants two weeks before the experiment to perform 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 indicators of transgenic lines and WT plants were evaluated. The evaluated growth traits include phenotype and plant biomass. Among them, the phenotype is recorded by taking photos using a digital camera; plant biomass is obtained from representative five seedlings (growth chamber culture) or twenty plants (field planting) after drying in an oven, using conventional methods. At maturity, the seeds are recorded for phenotype, and the grain weight is evaluated after seed air-drying, and the plot yield is based on 0.5 m 2 The air-dried grain weight of the plants was measured. The evaluated physiological indicators include indicators related to osmotic stress response, such as osmotic adjustment substance content, stomatal closure rate (SCR), in vitro leaf water loss rate (WLR), active oxygen-related parameters, using the upper expanded leaves as samples. Among them, the contents of osmotic adjustment substances proline and soluble sugar were evaluated (Zhao et al., 2024). The stomatal closure rate (SCR) value was determined according to the ratio of stomatal width observed at different drought time points [0.25, 0.5, and 1 hour during 1 hour drought treatment (10% PEG)] to 0 hour (Zhao et al., 2022). The in vitro leaf water loss rate (WLR) value was determined according to the reduction amount of fresh weight recorded at different time points (0.5, 1, and 3 hours) during drought treatment (leaves were placed on a clean table to start). Active oxygen-related indicators, such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, malondialdehyde (MDA) content, and active oxygen (i.e., superoxide anion and H2O2) accumulation were evaluated as follows: enzyme activity and MDA content (Huang et al., 2010) were determined; superoxide anion and H2O2 amounts were evaluated using histochemical staining methods, i.e., nitro blue tetrazolium (NBT) staining for superoxide anion and 3,3'-diaminobenzidine (DAB) staining for h2O2 (Zhao et al., 2024).

[0097] (14) Transcription activation experiment

[0098] Transcriptional activation assays of TaNF-YC1 on TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 in drought-stressed TaNF-YC1 transgenic lines in N. benthamiana expression system. For this, 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 long) of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 were amplified using wheat (Shimai 22) genomic DNA as template and gene-specific primers (Table 2) and integrated into the pGreenII 0800-LUC vector to generate the reporter constructs TaSLAC1-3pro::Luc, TaP5CR2::Luc, TaSOD4::Luc, TaCAT2::Luc and TaPIN6pro::Luc, respectively. The recombinant binary cassettes were co-transformed into Agrobacterium strain EHA105 at a 1:1 (v:v) ratio, combined as 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, CaMV35Spro::TaNF-YC1 and TaPIN6pro::LUC. The positive transformants were further used for transient transformation of N. benthamiana epidermal cells (Wang et al., 2020). After 48 h of transformation and 100 mM luciferin spray, the reporter luciferin signal in the transiently transformed cells was evaluated using a charge-coupled device imaging device (NightOWL II LB983 combined with Indigo software) following 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, i.e. TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6. For this, the ORF of TaPYR10 was amplified by RT-PCR using gene-specific primers (Table 2) and integrated into the EcoRI and Kpnl sites of the pGADT7 vector (Clontech) to produce the fusion protein pGADT7-TaPYR10 (as prey). Meanwhile, the promoter regions of its target genes were inserted into the Xhol and Kpnl sites of the pHIS2 reporter vector (Clontech) to produce the fusion proteins pHIS2-TaSLAC1-3, pHIS2-TaP5CR1, pHIS2-TaSOD4, pHIS2-TaCAT2 and pHIS2-TaPIN6 (as baits), respectively. The combinations consisting of prey and each bait plasmid were co-transformed into the yeast strain (EGY48). Positive transformants were identified after 3 days of incubation on solid selection growth medium lacking SD-Trp / -Ura. The results are shown in Figure 1.

[0101] (16) Statistical analysis

[0102] The mean values of the gene transcripts, growth traits, plant biomass and osmotic stress-related physiological indices were derived from three replicates. The standard error of the mean values and the statistical significance analysis of the traits were determined using the statistical analysis system software (SAS Corporation, Cary, NC, USA). The results of the regression analysis between the plant biomass and the expression levels of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 in the variety population under field drought conditions were evaluated using analysis of variance (ANOVA). The statistical significance analysis of the transgenic lines and the WT in the plant morphological traits and physiological indices was determined using Student's t-test (critical value P < 0.05). The heat maps showing the expression levels of TaPYR10 and stress-responsive genes and the yield of the wheat varieties in the field experiment were plotted using software called TBtools.

[0103] Example 2, Results

[0104] (1) Molecular characteristics of TaPYR10

[0105] The cDNA length of TaPYR10 is 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 shares high similarity with its counterparts from T. dicoccoides, A. tauschii, P. hallii, P. miliaceum, Z. mays, B. distachyon, S. bicolor, and O. brachyantha; they all contain a conserved PYR_PYL_PCAR domain composed of nine leaflets Figure 1 A).

[0107] At the nucleic acid level, TaPYR10 shares high identity with homologous genes in different plant species, with the highest similarity to genes from P. hallii, P. virgatum, S. viridis, Z. japonica, Z. mays, O. brachyantha, H. vulgare, A. tauschi, and T. dicoccoides Figure 2 ).

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

[0109] Transiently transformed cells expressing TaPYR10-GFP fusion proteins in the model plant N. benthamiana and wheat protoplasts showed that the GFP signal was localized to the nucleus Figure 1 C), which indicates that the TaPYR10 protein is localized to the nucleus after sorting through the endoplasmic reticulum (ER) and participates in biological functions.

[0110] (2) TaPYR10 expression sensitivity to drought signals

[0111] Analysis of TaPYR10 transcripts revealed changes in expression in response to drought and ABA signals. Under conditions of increasing drought, the expression levels of TaPYR10 in roots and leaves were significantly up-regulated, reaching the highest level at 15% PEG treatment Figure 3A). In addition, TaPYR10 transcripts in both tissues gradually increased during the 27-hour drought treatment and returned to normal levels after 27 hours of recovery conditions Figure 3 A). Similar TaPYR10 expression patterns were also observed in plant tissues treated with exogenous ABA; the transcripts of this wheat PYR gene were proven to be induced by ABA signals in a concentration- and time-dependent manner Figure 3 B). These findings suggest 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 DRE element that responds to osmotic stress, and the binding sites for transcription factors MYB and MYC that mediate gene transcription under stress Figure 3 C-D). These cis-regulatory elements are believed to play an important role in regulating TaPYR10 transcription under drought conditions. Under drought conditions, GUS staining and GUS activity were enhanced 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) compared to the control group (non-stress conditions) Figure 3 D-F). In addition, GUS staining and activity in leaves gradually increased as the promoter fragments were extended Figure 3 D-F). These results suggest that the DRE element and MYB and MYC transcription factor binding sites synergistically regulate TaPYR10 transcription 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 showed that the TaPYR10 protein can interact with the PP2C member TaPP2C30, which can interact with the SnRK2 member TaSnRK2.10. Similarly, the aforementioned SnRK2 member interacts with the NF-YC member TaNF-YC1 Figure 4 A). Based on the prediction results, experimental analysis was conducted on the interaction between TaPYR10 and its putative downstream partners. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), and in vitro pull-down experiments were used to study the role of TaPYR10 in establishing the ABA signaling module. In the yeast two-hybrid experiment, the TaPYR10 protein expressed in yeast cells (bait) specifically interacted with the PP2C member TaPP2C30 (prey) expressed in the yeast hostFigure 4 B). Likewise, TaPP2C30 protein was found to specifically interact 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 the yeast two-hybrid experiments were further confirmed by BiFC, clear YFP signals were detected in N. 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 pulldown assays of the above ABA signaling components verified the protein interaction results obtained from the yeast two-hybrid and BiFC experiments. Among them, Co-IP analysis showed that TaPYR10 could be co-immunoprecipitated with TaPP2C30, TaPP2C30 could be co-immunoprecipitated with TaSnRK2.10, and TaSnRK2.10 could be co-immunoprecipitated with TaNF-YC1 Figure 4 F-H). In vitro pulldown assays confirmed the protein interactions between the above ABA signaling partners Figure 4 I-K). These findings suggest 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 affecting drought responses.

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

[0115] To investigate the different regions involved in the protein-protein interactions between 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 the yeast two-hybrid experiment, positive yeast transformants were identified in cells co-expressing TaPYR10 middle fragment (TaPYR10-I1, amino acids 71-140, containing CL3-CL5 loops and PYR / PYL / PCAR conserved domain) and TaPP2C30 middle fragment (TaPP2C30-I1, amino acids 74-317, containing PP2C conserved activation domain) Figure 5B). Likewise, yeast host cells co-expressing TaPP2C30-I1 and the intermediate fragment of 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-terminal of TaNF-YC1 (TaNF-YC1-N1, amino acids 1 to 80, containing the nuclear localization signal) showed complementation growth ability on the selection medium SD / -Leu / -Trp lacking amino acids Figure 5 B). In yeast two-hybrid experiments, no interactions between TaPYR10 and other PP2C proteins, TaPP2C30 and other SnRK2 members, and TaSnRK2.10 and other NF-YC proteins were detected in addition to the previously described downstream partners of TaPYR10 Figure 6 ) BiFC experiments confirmed the interactions between these fragments in TaPYR10 and its downstream signaling partners. Reporter YFP signals were sensitively detected in N. 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 pulldown analyses confirmed the above-mentioned interaction processes obtained from yeast two-hybrid and BiFC experiments. Both experiments showed that TaPYR10 I1 co-immunoprecipitated with TaPP2C30 I1, TaPP2C30 I1 co-immunoprecipitated with TaSnRK2.10 I1, and TaSnRK2.10 I1 co-immunoprecipitated with TaNF-YC1 N1 Figure 5 D-I). These findings suggest that different protein regions in TaPYR10 and its downstream partners contribute to protein-protein interactions between members of the ABA signaling pathway.

[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 (Sen 2, Sen 3, Anti 1 and Anti 2), TaSnRK2.10 (Sen 1, Sen 2, Anti 2 and Anti 3) and TaNF-YC1 (Sen 2, Sen 3, Anti 2 and Anti 3) had significant changes in the expression of the target genes ( Figure 7 ), but were similar to wild type (WT) plants in growth traits, i.e. phenotype, 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 Sen 2), TaSnRK2.10 (Sen 1 and Sen 2) and TaNF-YC1 (Sen 2 and Sen 3) and the lines knocking down TaPP2C30 (Anti 1 and Anti 2) had increased phenotype ( Figure 8 A-D), improved biomass ( Figure 8 E-H), increased leaf area ( Figure 8 I-L), increased grain weight ( Figure 8 M-P) and improved yield ( Figure 8 Q-T) compared to wild type (WT) plants. In contrast, the lines knocking down TaPYR10 (Anti 1 and Anti 2), TaSnRK2.10 (Anti 2 and Anti 3) and TaNF-YC1 (Anti 2 and Anti 3) and the line overexpressing TaPP2C30 (Sen 2 and Sen 3) had deteriorated phenotype ( Figure 8 A-D), reduced plant biomass, leaf area, grain weight and yield relative to WT plants ( Figure 8 E-T). The growth characteristics 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 characteristics of these transgenic lines indicated that TaPYR10 and its signaling pathway genes play a key role in regulating plant drought and ABA response.

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

[0119] The physiological characteristics and indicators related to osmotic stress response in TaPYR10, TaPP2C30, TaSnRK2.10 and TaNF-YC1 lines were evaluated, including stomatal closure rate (SCR), in vitro leaf water loss rate (WLR), contents of osmotic adjustment substances proline and soluble sugar, photosynthetic parameters, active oxygen related indicators and root system morphological characteristics. Within 1 hour of drought treatment, the stomatal closure rate of TaPP2C30 knockdown (Anti 1 and Anti 2) and TaPYR10 (Sen 1 and Sen 2), TaSnRK2.10 (Sen 1 and Sen 2) and TaNF-YC1 (Sen 2 and Sen 3) overexpression lines increased compared with WT ( Figure 11 A-E). During 2 hours of leaf dehydration, the in vitro leaf water loss rate of these lines showed a downward trend compared with 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, and decreased non-photochemical quenching coefficient NPQ) Figure 12 ), and improved active oxygen related indicators, 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 reduced 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 overexpression (Sen 2 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 in vitro leaf water loss rate, decreased proline and soluble sugar contents, decreased photosynthetic capacity, deteriorated active oxygen homeostasis related indicators 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] A set of genes were subjected to expression analysis, including SLAC1 family genes (TaSLAC1-1 to TaSLAC1-6) that regulate stomatal movement, genes associated with proline accumulation (TaP5CS1, TaP5CS2, TaP5CR1, TaProDH1, TaP5CDH1), superoxide dismutase (SOD) family (TaSOD1 to TaSOD6) and catalase (CAT) family (TaCAT1 to TaCAT6) genes that mediate antioxidant enzyme activity, and PIN-FORMED (PIN) family genes (TaPIN1 to TaPIN6) that regulate root architecture establishment, which were analyzed in TaNF-YC1 transgenic lines after drought and exogenous ABA treatment. Among the genes tested, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 showed altered expression levels in TaNF-YC1 lines relative to WT plants (higher expression in Sen 2 and Sen 3, and lower expression levels in Anti 2 and Anti 3) Figure 16 A-E). In contrast, in TaNF-YC1 transgenic lines subjected to drought treatment, the transcripts of the other tested genes did not change compared to WT Figure 16 A-E). These results indicate that the differentially expressed genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 are affected at the transcriptional level by TaNF-YC1. Yeast one-hybrid experiments were performed to determine the protein / DNA interaction 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, 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 triple dropout medium (SD / -Leu-Trp-His) plates containing 45 mmol L -1 3-AT ( Figure 16G), indicating that TaNF-YC1 interacts with the promoters of stress-responsive genes. Transcription activation assays were performed in N. benthamiana to verify the regulation of TaNF-YC1 on the above-mentioned stress defense genes, and strong reporter gene LUC signals were detected in tobacco epidermal cells transiently co-transformed with the following plasmid combinations: effector cassette CaMV35S Pro::TaNF-YC1 and reporter cassette TaSLAC1-3 Pro::LUC Figure 16 H, M); effector cassette CaMV35S Pro::TaNF-YC1 and reporter cassette TaP5CR1 Pro::LUC Figure 16 I, N); effector cassette CaMV35S Pro::TaNF-YC1 and reporter cassette TaSOD4 Pro::LUC Figure 16 J, O); effector cassette CaMV35S Pro::TaNF-YC1 and reporter cassette TaCAT2 Pro::LUC Figure 16 K, P); and effector cassette CaMV35S Pro::TaNF-YC1 and reporter cassette TaPIN6 Pro::LUC Figure 16 L, Q). These results collectively 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) Differential osmotic stress-responsive gene positive regulation for drought stress adaptation

[0123] Transgenic analysis was performed on the differentially expressed stress-responsive genes TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6 to characterize their roles in mediating drought response. Under drought treatment, TaSLAC1-3 knockdown lines (AntiSLAC1-3A and AntiSLAC1-3B) had worse phenotypes, lower stomatal closure rates and increased transpiration rates Figure 17 A-C); TaP5CR1 expression knockdown lines (AntiP5CR1-1 and AntiP5CR1-2) grew slowly, had reduced biomass and proline content Figure 17 D-F); TaSOD4 knockdown (AntiSOD4-2 and AntiSOD4-3) resulted in phenotypic changes, reduced SOD activity and increased superoxide anion accumulation Figure 17 G-I, Figure 18 A); TaCAT2 knockdown lines (AntiCAT2-2 and AntiCAT1-3) had poor phenotypes, reduced CAT activity and increased H2O2 accumulation Figure 17 J-L, Figure 18B). TaPIN6 knock-down lines (AntiPIN6-1 and AntiPIN6-2) showed significantly reduced root growth, decreased root biomass, and reduced root volume Figure 17 M-O). Without drought treatment, these transgenic lines showed no significant difference in plant phenotype and root growth compared to WT plants. These results validated the positive effect of osmotic stress-related genes on drought adaptation at the transcriptional level of TaPYR10 module member TaNF-YC1.

[0124] (9) Transcript abundance of TaPYR10 and its downstream stress defense genes in wheat varieties under field drought conditions is highly correlated with yield

[0125] The expression levels of TaPYR10 and stress response genes (TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) were examined in a set of 45 wheat varieties with different drought tolerance performance in plant yield. Under field drought conditions, the transcripts of these genes in the upper leaves at the mid-grain filling stage showed significant differences Figure 19 A). Similarly, there were large differences in yield at maturity among the examined wheat varieties under field drought conditions 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 response genes Figure 19 C-H). These findings suggest that TaPYR10 and different stress defense genes act synergistically at the transcriptional level to regulate plant drought response and contribute to plant drought adaptation capacity.

[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 in the promoter region were examined by sequencing the ORF and flanking region (i.e., 2 kb upstream of the start codon) in 45 wheat varieties. Four SNPs in the ORF and five SNPs in the promoter region of TaPYR10 formed two major haplotypes, 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 for genotyping the 45 elite wheat varieties examined Figure 20B). 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 collectively indicate that TaPYR10-Hap1 has been positively selected in modern wheat breeding programs.

[0128] Example 3, Conclusion

[0129] (1) TaPYR10 response to drought signals is associated with specific cis-elements in the promoter

[0130] Expression of ABA signaling genes is tightly regulated at the transcriptional level. For example, transcripts of PYR family genes change in response to drought, salt, and ABA stress due to an increase in ABA levels in plant cells (Antoni et al., 2012; Li et al., 2018). Drought and exogenous ABA treatment also significantly induce the expression of PP2C genes regulated by different transcription factors such as 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 of transcription factors, GmNF-YC14, GmNF-YA16, and GmNF-YB2, activates the abscisic acid (ABA) signaling pathway mediated by GmPYR1, thereby regulating stress tolerance in 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 at the transcriptional level by drought and ABA signals.

[0131] Transcriptional changes of drought-responsive genes depend on cis-acting regulatory elements located on their promoters. Drought and ABA-responsive element (DRE), recognition sites for transcription factors MYB and MYC, and other elements play a key role in regulating gene drought response at the transcriptional level (Cutler et al., 2010; Singh and Laxmi, 2015). In this study, our analysis revealed a set of conserved elements in TaPYR10 promoter that control gene transcription, including TATA and CAAT boxes, and elements that trigger gene response to ABA and drought stress, i.e. DRE and recognition sites for MYB, MYC transcription factors. Their function in regulating gene response to drought and ABA signals was further validated by transforming wheat leaves with a series of truncated promoter fragments driving GUS expression, followed by GUS histochemical staining and activity assays. Additional experiments using base mutation approach can help determine the mechanism 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 ABA core signaling pathway. Functional modules of ABA signaling pathways involved in mediating ABA and drought responses have been established in plant species (Asad et al., 2019). Under stress conditions, accumulation of ABA and formation of PYR / PYL / RCAR-PP2C complex leads to PP2C inactivation, followed by SnRK2 activation. Activated SnRK2s promote transcription of ABA-responsive genes by phosphorylating downstream substrates (Ali et al., 2020). However, the downstream partners of ABA signaling members interacting with ABA receptors in cereal crops are 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 pulldown experiments, to determine the composition of the wheat ABA signaling pathway involving TaPYR10. Our results showed that TaPYR10 interacts with its downstream partners, 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 reported that ABA receptors can function in the nucleus (Cutler et al., 2010), and most PP2C family gene members were also found to be localized in the nucleus. Intracellular localization of ABA signaling partners contributes to the specificity of PP2C-RCAR pairing and their binding properties to downstream acting SnRK2s (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, suggesting that this can be the location where they exert their biological functions. To date, studies on different regions involved in protein-protein interactions between ABA receptors and their downstream partners are still limited. In this study, we determined the fragments involved in protein interactions between TaPYR10 protein and its downstream partners. Our results showed that the middle portion of TaPYR10 (amino acids 71-140), the middle portion of TaPP2C30 (amino acids 74-317), the middle portion of TaSnRK2.10 (amino acids 91-260) and the N-terminal of TaNF-YC1 (amino acids 1-80) contribute to protein interactions between TaPYR10 and its downstream partners. Further characterization of the fine positioning in these fragments that control the interactions can help to understand the molecular processes of protein interactions in the ABA signaling pathway in common wheat.

[0133] (3) TaPYR10 and its downstream partners have significant impacts on plant drought response by regulating stress-related physiological indicators. ABA receptor genes mediate plant drought and ABA responses through ABA-dependent signaling pathways (Park et al., 2009; González-Guzmán et al., 2014). Given their significant involvement in regulating stress defense physiological processes, including stomatal movement, osmoregulatory substance biosynthesis, cellular reactive oxygen homeostasis, and root 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 drought tolerance in poplar by regulating water use efficiency and reactive oxygen scavenging (Li et al., 2022). These studies have deepened our understanding of the mechanisms by which PYR1 / PYL / RCAR genes regulate plant drought adaptation. Similarly, studies on ABA receptor downstream genes have also shown that they play a key role in mediating drought response. The TaSnRK2.9 member of the SnRK2 family confers drought tolerance in transgenic tobacco plants (Feng et al., 2019). The MePP2C24 member of the PP2C family in cassava affects plant drought response through an ABA-dependent pathway (Zeng et al., 2024). In this study, we constructed transgenic lines of TaPYR10 and its downstream partners, TaPP2C30, TaSnRK2.10, and TaNF-YC1, including overexpression and knockdown expression lines, to characterize their functions in mediating drought response. 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 field condition drought treatment of TaPYR10 transgenic lines 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 response in this study confirmed their roles in regulating these physiological processes.

[0134] To characterize the molecular processes of TaPYR10 module in regulating the physiological processes related to osmotic stress, we analyzed the expression pattern of each genomic component to assess their 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 for proline biosynthesis (Bandurska et al., 2017), antioxidant enzymes (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 cell 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 to wild type, these genes had higher transcripts in transgenic TaNF-YC1 overexpression lines and lower transcripts in knockdown lines after drought treatment. These results suggest that these genes have potential roles in TaNF-YC1-regulated drought responses. We performed yeast one-hybrid experiments 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 interacts with the promoters of these genes by specifically binding to different promoter regions containing ABRE or MYB, which are cis-regulatory elements responsive to drought and ABA signals, respectively. Furthermore, our transcriptional activation experiments in tobacco (i.e., TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6) verified that they are all regulated by TaNF-YC1. In addition, 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.### TaPYR10 and expression changes in stress-responsive genes contribute to different drought tolerance in wheat varieties Genetic variation in wheat varieties is considered an essential element to enhance crop quantitative traits, including yield components and drought tolerance. Various approaches have been adopted to expand the genetic variation in crop plants, such as introduction of existing varieties, development of segregating material through local or international nurseries, hybridization, and mutagenic breeding (Thudi et al., 2021). Furthermore, the use of parental lines with different genetic backgrounds, including un-related and complementary genetic resources with suitable drought adaptation and yield-enhancing 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, namely 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, the expression levels of these genes in flag leaves were significantly different among varieties at the mid-grain filling stage. Furthermore, we found a high positive correlation between plant yield and the expression levels of TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6. In summary, these findings suggest that the genes TaPYR10, TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2, and TaPIN6 play a crucial role 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 to assess the drought adaptation capacity of wheat varieties grown under drought conditions. Based on the characterization of SNP behavior at the TaPYR10 promoter in the wheat variety panel, we revealed base variations in the sequence region flanking the MYB recognition site in the promoters of different drought-tolerant wheat varieties. Two haplotypes, including TaPYR10-Hap1 and TaPYR10-Hap2, were considered to affect plant drought responses by influencing the transcript abundance of the target genes. The haplotype TaPYR10-Hap1 confers better drought adaptation in wheat varieties, suggesting that this haplotype can 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, TaPYR10 transcripts are up-regulated under drought and ABA signal stimulation. The induced TaPYR10 protein interacts with the PP2C family member TaPP2C30 through a protein-protein interaction mechanism after binding to ABA molecules. The formed TaPYR10 / ABA / TaPP2C30 ternary complex eliminates the inhibition of TaPP2C30 on the common wheat SnRK2 family member TaSnRK2.10. Then TaSnRK2.10 interacts with the transcription factor member TaNF-YC1 of the wheat NF-Y family, and TaNF-YC1 participates in regulating osmolyte biosynthesis, stomatal movement and cellular reactive oxygen species homeostasis by activating the expression of TaSLAC1-3, TaP5CR1, TaSOD4, TaCAT2 and TaPIN6. The above-mentioned changed physiological processes help plants to adapt to drought stress. Further identification and functional analysis of the molecular processes of the signaling pathway in which TaPYR10 is involved can provide new insights into the plant drought tolerance mechanism.

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

[0136] In the above embodiments, the description of each embodiment is focused on, and the parts not described or recorded in detail in a certain embodiment can be referred to the relevant description of other embodiments.

[0137] The above-described embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be included in the protection scope of the present application.

Claims

1. A primer combination, characterized in that: This method is used to detect single nucleotide polymorphisms (SNPs) at the following SNP sites in the wheat genome. The physical distance between these SNP sites in the wheat genome version number Triticum aestivum iwgsc_refseqv1.0 is Chr1A_346681497. The four SNP sites in the ORF of TaPYR10 correspond to bases 211, 224, 374, and 410 of the sequence shown in SEQ ID NO.

1. When these sites are homozygous for G / G, T / T, G / G, and A / A in sequence, the corresponding genotype is Hap1. When the sites are homozygous for T / T, A / A, C / C, and G / G, the corresponding genotype is Hap2. The five SNP sites in the promoter region correspond to SEQ ID NO. In the sequence shown in .9, the 1680th, 1684th, 1685th, 1737th, and 1902nd bases from the end of the sequence, when these sites are homozygous for T / T, A / A, C / C, A / A, and G / G respectively, correspond to the genotype Hap1. When the sites are homozygous for C / C, G / G, T / T, G / G, and T / T respectively, the corresponding genotype is Hap2. The proline content, biomass, and yield are as follows: wheat homozygous for genotype Hap1 is greater than or candidate greater than wheat homozygous for genotype Hap2. The primer combination is the primer pair composed of SEQ ID NO.244 ~ SEQ ID NO.279 in the sequence listing.

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