CN120173966B - Functional module for regulating drought response of plants and application thereof - Google Patents

Abstract

This invention discloses a functional module for regulating plant drought response and its applications, the module comprising TaPYR10 and its downstream signaling pathway members. By revealing the roles of TaPYR10 and its downstream partners (TaPP2C30, TaSnRK2.10, and TaNF-YC1) in drought signal transduction, this invention provides a new molecular basis for wheat drought tolerance. Functional analysis of these genes indicates that they play a crucial role in plant responses to drought stress, enhancing plant resistance by regulating related physiological processes such as stomatal movement, synthesis of osmotic regulators, and cellular reactive oxygen species homeostasis.

Description

Functional module for regulating drought response of plants and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a functional module for regulating drought response of plants and application thereof.

Background

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

Numerous studies have shown that drought stress has a significant impact on physiological, biochemical and molecular processes that affect moisture relationships at the cellular, tissue and organ level. For major crops, drought results in significant reductions in relative water content, chlorophyll content, and plant productivity. Thus, plants have evolved a variety of strategies to mitigate the negative effects of drought, enhancing their osmotic regulation by accumulating osmotic regulating substances (such as proline, soluble sugars, betaines, organic acids, free amino acids and other organic compounds) to maintain adequate moisture supply. The plant can also activate antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and Catalase (CAT) to increase active oxygen scavenging ability and maintain active oxygen homeostasis. In addition, plant hormones such as auxin (IAA), gibberellin (GA), zeatin nucleoside (ZR), abscisic acid (ABA) and Jasmonic Acid (JA) act synergistically to mediate plant drought responses.

Abscisic acid (ABA) is a major stress-responsive hormone produced by plants in response to drought, and it affects multiple physiological processes throughout the life cycle of the plant, including seed dormancy and germination, root growth, stomatal guard cell expansion, senescence, and fruit ripening. ABA mediated stress responses are achieved through specific ABA signaling pathways in which plant cells sense ABA signaling and convert it into a variety of physiological and biochemical responses through signaling processes. When plants are exposed to drought conditions, members of the ABA receptor family (i.e., PYR/PYL/RCAR) play a role in sensing drought signals in the environment and triggering ABA signal transduction through protein phosphorylation mechanisms. PYR/PYL/RCAR members, type 2C protein phosphatase (PP 2C) proteins, and SNF 1-related protein kinase 2 (SnRK 2) family members have been shown to be core signaling components of the drought-induced ABA-dependent pathway. Recent studies have established a working model of ABA signaling pathways. Under conditions of sufficient moisture and low ABA levels, the activity of SnRK2 members is inhibited by physical interactions with the type A PP2C (PP 2C-A) protein, which dephosphorylates multiple serine/threonine residues located in the activation loop. This inhibition inhibits the physiological, biochemical and molecular processes involved in plant drought response. In addition, under drought conditions, ABA concentration in the tissue increases and then binds to PYR/PYL/RCAR proteins, allowing ABA receptors to interact with PP2C proteins, resulting in release of SnRK2 from inhibition of PP2C, activating downstream targets such as ABA response element binding factor (ABFs), ABA response element binding protein (AREBs), and transcription factor members of NAC, MYC, and MYB. Thus, ABA receptor mediated signaling pathways are key regulators of plant drought response.

Recent studies identified and characterized NF-Y family members and PP2C and SnRK2 family members in the model plant arabidopsis. Functional characterization of NF-Y genes suggests that they play a key role as mediators of plant drought stress response, as they regulate biological processes associated with drought stress response. Among NF-Y genes in maize, zmNF-YA1 and ZmNF-YB16 have been demonstrated to confer better drought adaptation to plants by enhancing transcription of osmotic stress response genes and improving water use efficiency of plants under drought stress. The ABA receptor family member PYR/PYL/RCAR is located downstream of NF-Y transcription factors, of which TaPYL has been demonstrated to up-regulate the expression of ABA biosynthesis genes (including ZEP1, NCED2, NCED3 and NCED 4), which helps to maintain cell water content, membrane stability, chlorophyll biosynthesis, reduce active oxygen (e.g. h ₂O₂) levels and increase plant productivity under drought conditions. In arabidopsis, ten SnRK2 family members were identified, and expression analysis showed that all SnRK2 members in arabidopsis, except SnRK2.9, were activated by osmotic stress, and functional characterization demonstrated that they were involved in transducing osmotic stress signals through ABA-dependent pathways. In addition, the type a PP2C protein in arabidopsis plays a critical role as a negative regulator in the ABA signaling pathway. Thus, the different members of the ABA receptor family constitute signaling pathways with downstream partners, regulating stress defense biological processes primarily through ABA-dependent pathways, contributing to plant drought tolerance.

Wheat is an important crop that is widely planted worldwide. With the increase of global warming, soil drought is increasingly serious, and the wheat yield is negatively affected. Although ABA receptors and their downstream partners (such as PP2C protein and SnRK2 members) have been extensively characterized in model plant species and in some crop species, the functional characterization of ABA receptor family members and their downstream partners in PP2C and SnRK2 families, as well as transcription factor members involved in plant drought defence, is still limited in common wheat.

Disclosure of Invention

The invention aims to provide a functional module for regulating drought response of plants and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows.

A functional module for modulating drought response in a plant comprising TaPYR a10 and downstream signal pathway members thereof.

Further preferred, the downstream signal pathway members include TaPP C30, taSnRK2.10, and TaNF-YC1.

A method for cultivating drought-resistant transgenic plant includes such steps as editing TaPYR gene and downstream signal channel members, and cultivating the transgenic plant with drought-resistant characteristics.

Further preferred are, in particular, overexpression TaPYR, taSnRK2.10 and TaNF-YC1 genes, suppression or reduction or silencing or knocking out of the TaPP C30 gene.

The kit is used for regulating the drought response capacity of plants, and comprises a molecular biological element capable of regulating and controlling the expression quantity of specific genes, wherein the specific genes are genes related to the drought response capacity regulation of the plants.

Further preferred, the molecular biological elements are a combination of elements that overexpress TaPYR10, tasnrk2.10, and the TaNF-YC1 gene and that inhibit or reduce or silence or knock out the TaPP C30 gene.

A method of enhancing drought stress tolerance in a plant by overexpressing TaPYR, tasnrk2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out TaPP C30 genes to enhance drought stress tolerance in a plant.

A method for increasing plant yield by overexpressing TaPYR, tasnrk2.10 and TaNF-YC1 genes and inhibiting or reducing or silencing or knocking out TaPP C30 genes to enhance drought stress tolerance of the corresponding plants, thereby increasing yield of the corresponding plants.

According to the functional module or the method or the kit or the method, the plant is wheat.

The application of the functional module comprising TaPYR10 and downstream signal path members thereof evaluates the drought response capability of plants by analyzing the expression level of TaPYR and downstream signal modules thereof, screens, preliminary screens, identifies and assists in identifying wheat germplasm resources with strong drought resistance capability, in particular screening, preliminary screens, identifies and assists in identifying wheat germplasm which overexpresses TaPYR, taSnRK2.10 and TaNF-YC1 genes and inhibits or reduces or silences or knocks out TaPP C30 genes.

A method for enhancing drought stress tolerance of wheat comprises introducing multiple copies of cis-element DRE and MYB and MYC TF protein binding sites into wheat genome by gene editing technology, and constructing a system capable of over-expressing TaPYR under drought and ABA signal stimulation to enhance drought stress tolerance of wheat.

A SNP locus related to the proline content, biomass and yield of wheat is characterized in that the SNP locus is positioned on chromosome 1 of wheat, the physical distance of the SNP locus in the genome version number Triticum aestivum iwgsc-refseqv.0 of the wheat is Ch1A_ 346681497, 4 SNP loci of 0RF in TaPYR10 correspond to bases 211, 224, 374 and 410 of the sequence shown in SEQ ID NO.1 respectively, the loci are G/G, T/T, G/G, A/A homozygotes in sequence, the corresponding genotypes are Hap1, the genotypes are Hap2 when the loci are T/T, A/A, C/C, G/G homozygotes, 5 SNP loci in the promoter region correspond to bases 1680, 1684, 1737 and 1902 of the sequence shown in SEQ ID NO.9 from the last reciprocal, the genotypes are T/T, A/A, C/3696/3742/G homozygote, the genotypes are Hap1, the genotypes are Hap2 when the genotypes are T/T, A/A, C/C, G/G homozygote, the genotypes are expressed in sequence shown in SEQ ID NO.9, and the gene quantities are expressed in sequence from the last to be the last to the last nucleotide of 1680, the nucleotide of the nucleotide sequence shown in SEQ ID NO.1, the nucleotide sequence is the nucleotide sequence of SEQ ID NO. is the nucleotide 1, the nucleotide 1 is the nucleotide 1, the nucleotide sequence is the nucleotide 1 is the nucleotide sequence, and the nucleotide sequence is the nucleotide sequence corresponding to the nucleotide sequence, and the nucleotide sequence is the nucleotide sequence 3 is the nucleotide.

A primer combination is characterized by being used for detecting single nucleotide polymorphism of SNP sites in a wheat genome, wherein the physical distance between the SNP sites in the wheat genome version number Triticum aestivum iwgsc-refseqv and 1.0 is Ch1A_ 346681497, 4 SNP sites of 0RF in TaPYR10 correspond to bases 211, 224, 374 and 410 of a sequence shown in SEQ ID NO.1 respectively, the corresponding genotypes are Hap1 when the sequences are G/G, T/T, G/G, A/A homozygotes, the corresponding genotypes are Hap2 when the sequences are T/T, A/A, C/C, G/G homozygotes, 5 SNP sites in the promoter region correspond to bases 1680, 1685, 1737 and 1902 of the sequence shown in SEQ ID NO.9 respectively, the corresponding genotypes are T/T, A/A, C/42/3625/G homozygotes when the sequences are G/G, T/T, G/G, A/A homozygotes, the corresponding genotypes are Hap1, the corresponding genotypes are Hap2 when the sequences are T/T, A/A, C/C, G/G homozygotes, and the gene sequences shown in the promoter region correspond to the sequence shown in SEQ ID NO.9, and the gene combination is larger than the gene sequence of the gene pairs of the sequence shown in SEQ ID NO.1 and the gene combination is used for detecting the gene sequence of the sequence from the sequence of the sequence 1 to the sequence.

According to the SNP locus, kasp molecular markers related to drought resistance of wheat are developed.

A method for culturing wheat with drought tolerance and high yield includes such steps as early breeding, detecting the transcript abundance of TaPYR gene and stress-protecting genes TaSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN6, selecting the individuals whose transcript abundance of TaPYR gene and stress-protecting gene is higher than the predefined threshold value, collecting the wheat varieties with different drought tolerance and yield, testing their transcript abundance in upper leaf in medium-filling stage and yield data in mature stage, statistical analysis, culturing the screened individuals to obtain wheat variety or strain with high drought tolerance and high yield, and greatly increasing the breeding efficiency and shortening the period.

The beneficial effect produced by adopting the technical scheme is that in the research, we concentrate on characterizing the ABA receptor gene TaPYR in wheat to identify the downstream partner and functionally characterize the ABA core signal module in mediating the drought response of plants. Our research results provide new insight for understanding the mechanism of ABA signaling pathway in plant drought response, and may promote development of drought-tolerant varieties of common wheat by molecular breeding;

The invention provides a new molecular basis for drought tolerance of wheat by revealing the role of TaPYR and downstream partners (TaPP C30, taSnRK2.10 and TaNF-YC 1) thereof in drought signal transduction. Functional analysis of these genes shows that they play a key role in plant response to drought stress, and can improve stress resistance of plants by regulating related physiological processes (such as stomatal movement, synthesis of osmoregulation substances and cellular reactive oxygen species homeostasis);

According to the invention, through analysis of cis-regulatory elements in TaPYR promoter, research reveals that the expression is precisely regulated by drought and ABA signals, thus providing new insight for understanding the gene regulation mechanism of plants under environmental stress, providing new targets for genetic engineering and breeding, and improving breeding efficiency;

The invention verifies the functions of TaPYR and downstream partners thereof in drought response by constructing the transgenic wheat strain, provides genetic resources for subsequent crop improvement, and has important application prospect in the aspect of improving wheat drought tolerance.

Drawings

FIG. 1 is a schematic diagram of a characterization of TaPYR proteins, wherein A is the TaPYR protein and its counterparts in various plant species, B is the simulated three-dimensional structure of the TaPYR protein, C is the fluorescent signal contrast of TaPYR-GFP with GFP in N.benthamiana and wheat protoplasts, the conserved domains designated by I through IX are highlighted in A and B, and the arrows point to the nuclei in C;

FIG. 2 is a schematic representation of the phylogenetic relationship at the nucleic acid level of TaPYR and its plant homologues;

FIG. 3 is a schematic representation of the expression pattern of TaPYR10 under drought and ABA treatment, wherein panels A-B are schematic representations of the expression pattern in roots and leaves under TaPYR drought treatment (panel A) and under ABA treatment (panel B), 0, 1%, 5%, 10% and 15% represent the concentration of PEG (w/v) in panel A, 1h, 3h, 9h and 27h represent the time after drought treatment (10% PEG, w/v), R1h, R3h, R9h and R27h represent the time to recover treatment after 27h drought challenge, 0h represent the time points before drought treatment, 0, 0.5. Mu. Mol, 1.5. Mu. Mol and 2. Mu. Mol represent the concentration of exogenous ABA in panel B, 1h, 3h, 9h and 27h represent the time after ABA treatment (1.5. Mu. MolABA), panel C is a binary expression cassette for TaPYR promoter integration, panel D is a schematic representation showing cis-element associated with osmotic stress, R1h, R3h, R9h and R27h represent the time to recover treatment after 27h drought challenge, 0h represents the time point before drought treatment, 0.5. Mu. Mol, 1.5. Mu. Mol and 2. Mu. Mol represent the concentration of exogenous ABA in the plant (35 bp) and 35 bp) in the map B, 35 bp 10, 35 bp promoter 10-35, 35 bp 10% and 37 bp gene 35F (35% in the map B) are the same gene map 35, 35 bp, and 35 bp map 35B, and the map 35 gene 35;

FIG. 4 is a schematic representation of the protein interaction assay of TaPYR protein with a downstream partner, wherein Panel A is a schematic representation showing predicted interactions between TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 protein, panel B-D is a schematic representation of the yeast two-hybrid assay showing interactions between TaPYR and TaPP C30 (Panel B), taPP C30 and TaSnRK2.10 (Panel C) and TaSnRK2.10 and TaNF-YC1 (Panel D), panel E is a schematic representation of the BiFC assay verifying interactions between TaPYR C10 and its downstream partner, and Panel F-H is a schematic representation of the immunoprecipitation assay showing interactions between TaPYR and TaPP C30 (Panel F), taPP C30 and TaSnRK2.10 (Panel G) and between TaSnRK2.10 and TaNF-YC1 (Panel H), and Panel I-K is a pull-down assay showing interactions between TaPYR and TaPP C30 (Panel I), taPP C2C 30 and TaSnRK2.10 (Panel H) and TaSnRK2.10 (Panel J);

FIG. 5 is a schematic representation of experimental results of protein fragments of TaPYR10 and its downstream partners; wherein panel a is a schematic representation of individual fragments of TaPYR10, taPP2C30, tasnrk2.10 and TaNF-YC1 proteins, which fragments were used for yeast two-hybrid and BiFC experiments; FIG. B is a schematic representation of the results of a yeast two-hybrid experiment, showing the contributions of different fragments of TaPYR10, taPP2C30, taSnRK2.10 and TaNF-YC1 proteins to protein interactions; FIG. C is a schematic representation of BiFC experiments demonstrating fragments involved in interactions between TaPYR10, taPP C30, taSnRK2.10 and TaNF-YC1 proteins, in FIGS. B and C TaPYR10I1 represents an intermediate fragment of TaPYR (amino acids 44-192), taPP C30I1 represents an intermediate fragment of TaPP2C30 (amino acids 74-317), taSnRK2.10I1 represents an intermediate fragment of TaSnRK2.10 (amino acids 22-278), taNF-YC1N1 represents the N-terminus of TaNF-YC1 (amino acids 81-255), FIGS. D-F are schematic representations of immunoprecipitation experiments demonstrating the interactions of proteins between TaPYR (TaPYR I1) and TaPP C30 (TaPP C30I 1), the interactions between 8628C 30 (TaPP C30I 1) and TaSnRK2.10 (TaSnRK2.10I1), and the interactions between TaNF-YC1N1 (FIG. 10) and TaNF 2.10I1 (35I 1), and the interactions between TaNF-YC1 (35I 1.10F 1 and TaNF-YC1 (35I 1) and the in vitro interactions between TaRK2.10F 1 and TaRK1 (35I 1.10I 1) and TaNF-YC1 (35I 1) and FIG. 3I 1, FIG. 3.F is shown as schematic representation of the interactions between TaRK2I 1 and TaFC 1F 1;

FIG. 6 is a schematic diagram of experimental results of assessing TaPYR interactions between PP2C, snRK2 and NF-YC family proteins by a yeast two-hybrid method, wherein FIG. A is a yeast two-hybrid experimental result between TaPYR and a PP2C family member, FIG. B is a yeast two-hybrid experimental result between TaPP C2C 30 and a SnRK2 family member, and FIG. C is a yeast two-hybrid experimental result between TaSnRK2.10 and a NF-YC family member;

FIG. 7 is a schematic representation of target gene expression levels of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 in transgenic wheat lines, wherein graphs A-D are expression levels of the target gene in the transgenic lines A being TaPYR transgenic line, B being TaPP C30 transgenic line, C being TaSnRK2.10 transgenic line, D being TaNF-YC1 transgenic line, expression values being normalized by Tatubulin and Taactin, expression levels of both being set to 1, data being mean.+ -. Standard deviation (n=3), different lower case letters representing significant differences between transgenic lines and Wild Type (WT) (Tukey test, P < 0.05);

FIG. 8 is a schematic representation of phenotype and growth characteristics of a transgenic plant line of TaPYR and its downstream partner genes under drought treatment, wherein panels A-D are schematic representation of phenotype results of the medium filling period under drought treatment, panels E-H are schematic representation of plant biomass results of the medium filling period when grown in the field, panels I-L are schematic representation of leaf area results of the medium filling period plants under field conditions, panels M-P are schematic representation of grain weight results of plants cultivated under field conditions, panels Q-T are schematic representation of yield results of plants grown under field conditions, soil moisture content under normal conditions is 70% -75% of field water retention in panels E-T, drought treatment, soil moisture is 55% -60% of field water retention in panels A-T, data are expressed as mean.+ -. Standard deviation (n=3), statistical significance between transgenic plant and plant under the same growth conditions using Student's T-test (< P < 0.05);

FIG. 9 is a schematic representation of plant phenotypes of the TaPYR, taPP2C30, taSnRK2.10 and TaNF-YC1 transgenic lines (overexpressed or knocked down) grown under normal conditions in the field;

FIG. 10 is a schematic representation of phenotype and plant biomass of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 transgenic lines under exogenous ABA treatment, wherein FIG. A is a schematic representation of phenotype results of TaPYR transgenic lines, FIG. B is a schematic representation of phenotype results of TaPP C30 transgenic lines, FIG. C is a schematic representation of phenotype results of TaSnRK2.10 transgenic lines, FIG. D is a schematic representation of phenotype results of TaNF-YC1 transgenic lines, FIG. E is a schematic representation of plant biomass results of TaPYR10 transgenic lines, FIG. F is a schematic representation of plant biomass results of TaPP C30 transgenic lines, FIG. G is a schematic representation of plant biomass results of TaSnRK2.10 transgenic lines, FIG. H is a schematic representation of plant biomass results of TaNF-YC1 transgenic lines, exogenous ABA (1. Mu. Mol) is sprayed in the second leaf stage, and data are expressed as standard deviations of + -.standard deviations (n=3) in FIG. E. Statistical significance between transgenic lines and WTs was tested using Student's t-test (< 0.05 p and <0.01 p);

FIG. 11 is a graph showing osmotic stress related physiological characteristics of TaPYR and its downstream partner transgenic lines, wherein graph A shows the results of stomatal characteristics under drought stress, graph B-E shows the results of stomatal closure rate during 2 hours drought treatment, graph F-I shows the results of leaf moisture loss during 2 hours moisture loss, graph J-M shows the results of proline content, and graph N-Q shows the results of soluble sugar content, in graph B-Q, data are expressed as mean.+ -. Standard deviation (n=3), statistical significance between transgenic lines and WT under similar conditions using Student's t-test (< 0.05);

FIG. 12 is a graph showing the results of photosynthetic parameters of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment, wherein graphs A-D are graphs showing the net photosynthetic rate (Pn) results, graphs E-H are graphs showing the actual quantum efficiency (ΦPSII) results of photosystem II, graphs I-L are graphs showing the stomatal conductance (Gs) results, graphs M-P are non-photochemical quenching coefficients (NPQ), data are expressed as mean.+ -. Standard deviation (n=3), and different lower case letters indicate significant differences between transgenic lines at the same growth stage (Tukey test, P < 0.05);

FIG. 13 is a graph showing ROS-related index and root phenotype of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment, wherein graphs A-D are graphs showing the results of superoxide anion content assessed by nitrosoblue tetrazolium (NBT) staining, graphs E-H are graphs showing the results of H ₂O₂ content assessed by 3, 3-Diaminobenzimidazole (DAB) staining, graphs I-L are graphs showing the results of superoxide dismutase (SOD) activity, graphs M-P are graphs showing the results of Catalase (CAT) activity, graphs Q-T are graphs showing the results of crude enzyme (POD) activity, graphs U-X are graphs showing the results of root phenotype, data in graphs I-T are expressed as mean.+ -. Standard deviation (n=3), statistical significance between WT and transgenic lines was tested using Student's T-test (< 0.05);

FIG. 14 is a graphical representation of the results of the superoxide anion and H ₂O₂ content of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment, wherein graphs A-D are graphical representations of the results of the superoxide anion content in transgenic lines assessed by nitrosoblue tetrazolium (NBT) staining, and graphs E-H are graphical representations of the results of the H ₂O₂ content in transgenic lines assessed by 3, 3-Diaminobenzimidazole (DAB) staining;

FIG. 15 is a graph showing MDA content and root characterization results of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 transgenic lines under drought treatment, graphs A-D are graphs showing MDA content results, graphs E-H are graphs showing root biomass results, graphs I-L are graphs showing root volume results, data are expressed as mean value.+ -. Standard deviation (n=3), and different lower case letters indicate significant differences between transgenic lines under the same growth conditions (Tukey test, P < 0.05);

FIG. 16 is a graph showing results of expression of stress-related genes, yeast single hybridization and transcriptional activation characteristics, wherein graphs A to E are graphs showing expression of SLAC1 gene (graph A), proline accumulation-related gene (graph B), SOD gene (graph C), CAT gene (graph D) and PIN gene (graph E) in TaNF-YC1 over-expressed lines under drought treatment; FIG. F is a schematic representation of cis-acting elements located in the promoters TaSLAC-3, taP-5 CR1, taSOD4, taCAT and TaPIN (CACGTG, ACGTG, AACCCGG, GACACGTGGC, CGCACGTGTC), the ABRE, ABA reaction element (CACGTG, ACGTG, AACCCGG, GACACGTGGC, CGCACGTGTC), MYB, drought reaction element (CAACCA, CAACAG, TAACCA), FIG. G is a schematic representation of yeast single hybridization assay results showing normal growth of pGADT7-TaNF-YC1 and pHIS 2-TaSLAC-3 Pro, pGADT7-TaNF-YC1 and pHIS2-TaP CR1Pro, pGADT7-TaNF-YC1 and pHIS2-TaSOD4Pro, pGADT7-TaNF-YC1 and pHIS2-TaCAT Pro, pGADT7-TaNF-YC1 and pHIS2-TaPIN Pro in 45mM3-AT containing deletion medium (SD/-Leu-Trp-His) plates, and that shows normal growth of pGADT7-TaNF-YC1 and pHIS2-TaPIN Pro in N.Tantamia cells, and that pGADT7-TaNF-YC1 and pHIS2-TaCAT Pro (FIG. 1:, taCAT2Pro:: LUC (panel K) and TaPIN6Pro:: LUC (panel L) followed by a graph of the detected fluorescein signal, panel M-Q is a graph of the results of LUC activity in leaves co-transformed with the effector and reporter combinations mentioned in panel H-L, panel M-Q data expressed as mean.+ -. Standard deviation (n=3), statistical significance between LUC activity using Student's t-test to test the effector/reporter (p < 0.05);

FIG. 17 is a graph showing the results of physiological index and root morphology of a knockdown strain under drought treatment, wherein graphs A-C show the results of stomatal morphology (graph A), stomatal opening (graph B) and transpiration rate (graph C) of a TaSLAC1-3 knockdown strain during drought treatment for 1 hour, and graphs AntiSLAC1-3A and AntiSLAC1-3B show TaSLAC1-3 knockdown strain; panels D-F are graphs showing phenotype (panel D), plant biomass (panel E) and proline content (panel F) results for the TaP5CR1 knockdown strain at the seedling stage, antiP CR1-1 and AntiP CR1-2 are graphs showing the results for the TaP5CR1 knockdown strain, panel G-I is a graph showing phenotype (panel G), superoxide dismutase (SOD) activity (panel H) and histochemical staining for the TaSOD4 knockdown strain at the seedling stage to show accumulated superoxide anion (panel I) results, antiSOD4-2 and AntiSOD4-3 are graphs showing TaSOD4 knockdown strain, panel J-L is a graph showing phenotype (panel J) for the TaCAT2 knockdown strain at the seedling stage, catalase (CAT) activity (panel K) and histochemical staining to show accumulated H ₂O₂ (panel L) results, panels AntiCAT-2 and AntiCAT-2 are graphs showing TaCAT knockdown strain, panel M-O is a graph showing the results for the TaPIN knockdown strain cultured under field conditions, panel M4-2 and AntiSOD-3 is a graph showing the average root volume (panel N) and the average value (panel N6-6) is a graph (panel N=6-6 and the average value (panel N=6-3) is a graph of 37-2 and the average value (panel N=6-6 and the average value is shown in graph K-6-K is a graph 1-K), the transgenic lines were tested for statistical significance with wild type under the same growth conditions using Student's t-test (< 0.05). ;

FIG. 18 is a graph showing the results of the levels of superoxide anions and H ₂O₂ in TaSOD4 and TaCAT knockout lines under drought treatment;

FIG. 19 is a graph showing the transcriptional characteristics of TaPYR and its downstream stress defense genes and crop yield characterization in wheat varieties under drought conditions in the field, wherein graph A shows the results of the expression levels of TaPYR, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 in the tested wheat varieties, graph B shows the results of the yields in the tested wheat varieties, graph C-H shows the results of the regression analysis of the yields with TaPYR10 transcript (graph C), the results of the regression analysis of the yields with TaSLAC1-3 transcript (graph D), the results of the regression analysis of the yields with TaP5CR1 transcript (graph E), the results of the regression analysis of the yields with TaSOD4 transcript (graph F), the results of the regression analysis of the yields with TaCAT2 transcript (graph G), and the results of the regression analysis of the yields with TaPIN6 transcript (graph H), and 45 tested wheat varieties on the left side of the graph A-B are simplified according to the registered names;

FIG. 20 is a schematic representation of the effect of TaPYR haplotypes on proline, plant biomass and yield traits, wherein FIG. A is a schematic representation of the alignment of TaPYR haplotypes Hap1 and Hap2 and their deduced amino acid sequences, the red displayed polymorphic sites are used to develop a haplotype specific marker for TaPYR, FIG. B is a representation of genotyping a panel of wheat varieties using the TaPYR haplotype specific marker, FIG. C is a haplotype heat map cluster based on genes associated with TaPYR, and FIGS. D, E, F are schematic representation of yield, plant biomass and proline results between wheat varieties, respectively, of the alignment haplotypes Hap1 and Hap 2;

FIG. 21 is a schematic diagram of a working model of TaPYR and its downstream partners in mediating plant drought responses.

Detailed Description

The following examples illustrate the invention in detail. The raw materials and the equipment used by the invention are conventional commercial products, and can be directly obtained through market purchase. The experimental methods used in the following examples are conventional methods unless otherwise specified.

It should be understood that the terms "comprises" and/or "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It should also be understood that the term "and/or" as used in the present specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations.

Reference in the specification to "one embodiment" or "some embodiments" or the like means that a particular feature, structure, or characteristic described in connection with the embodiment is included in one or more embodiments of the application. Thus, appearances of the phrases "in one embodiment," "in some embodiments," "in other embodiments," and the like in the specification are not necessarily all referring to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "comprising," "including," "having," and variations thereof mean "including but not limited to," unless expressly specified otherwise.

Furthermore, the terms "first," "second," "third," and the like in the description of the present specification and in the appended claims, are used for distinguishing between descriptions and not necessarily for indicating or implying a relative importance.

The following description of the present invention will be made more complete and clear in view of the detailed description of the invention, which is to be taken in conjunction with the accompanying drawings that illustrate only some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1, materials and methods

(1) Plant material and growth conditions wheat (Dan Mai) seedlings hydroponic in a growth chamber were used to analyze TaPYR expression under drought and ABA treatment. TaPYR (nucleotide sequence shown as SEQ ID NO. 1), its downstream partner TaPP C30 (nucleotide sequence shown as SEQ ID NO.3, amino acid sequence shown as SEQ ID NO. 4), taSnRK2.10 (nucleotide sequence shown as SEQ ID NO.5, amino acid sequence shown as SEQ ID NO. 6), taNF-YC1 (nucleotide sequence shown as SEQ ID NO.7, amino acid sequence shown as SEQ ID NO. 8) and seedlings of the transgenic lines regulated by TaNF-YC1 were cultivated in plastic pots containing soil (half vermiculite and half fertile soil) in the growth chamber to determine the function of the gene in mediating drought and ABA responses in plants.

Wheat seedlings were grown at 18-22℃with 16 hours of light/8 hours of dark cycle and supplemented with light provided by high pressure sodium lamps (Powertone SON-TAGRO 400W;Philips Electronic UKLtd,Farnborough,UK).

The above transgenic lines and core wheat variety populations including 45 varieties were evaluated for growth traits and gene expression levels in field experiments conducted at the institute of food and oil crop research stations (37 ° 56'24.62″ north latitude, 114 ° 42'46.96″ east longitude) at the institute of agriculture and forestry science, national institute of northwest, rocky home, northwest, china in 2022-2023 and 2023-2024 planting season.

TABLE 1 core wheat variety population information for 45 varieties participating in field trials

(2) Molecular characterization

The molecular properties of wheat ABA receptor (PYR) family member TaPYR (GenBank accession number TraesCS A02G 191700) were characterized by retrieving the Open Reading Frame (ORF) of TaPYR and its 2kb promoter region (nucleotide sequence shown in SEQ ID NO. 9) from Ensembl Plants website (www.http:// Plants. Ensembl. Org/index. Html). Definitions TaPYR conserved domains of proteins similar to other PYR counterparts. By BLASTN search analysis of the GenBank database of the national center for Biotechnology information (NCBI, https:// www.blast.ncbi.nlm.nih.gov/blast. Cgi), phylogenetic relationships between TaPYR and its homologous genes in different plant species were obtained and their associations were established using the MegAlign algorithm in DNAStar software (https:// www.dnastar.com). ClustalW alignment was performed on the TaPYR protein and its plant counterparts using MEGA11 software (https:// www.mega.com). The three-dimensional structure of TaPYR proteins was modeled using the on-line tool SWISS-MODEL algorithm (https:// swissmodel. Expasy org/interactive), and the relevant prediction program was used as suggested.

(3) Protein subcellular localization analysis

Subcellular localization of TaPYR protein was determined by detecting the signal of TaPYR-GFP (green fluorescent protein, GFP) in the epidermal cells of Nicotiana benthamiana. For this purpose, the ORF of TaPYR was amplified based on reverse transcription polymerase chain reaction (RT-PCR) using gene specific primers and integrated with a reporter Gene (GFP) in frame under the control of the CaMV35S promoter into binary vector pCAMBIA 3300. The resulting expression cassette TaPYR-GFP was then transformed into Agrobacterium (strain EHA 105) using conventional heat shock methods. Positive transformants were used for transient transformation of tobacco surface cells of benthamia using agrobacterium-mediated transformation methods (Li et al 2012). After 48 hours of transformation, GFP signals were observed in cells containing TaPYR-GFP fusion constructs and cells integrated with empty vector using fluorescence microscopy.

TABLE 2 Gene-specific primers

(4) TaPYR10 expression analysis

Trefoil wheat (variety Dan Mai) seedlings grown in modified Murashige-Skoog (MS) solution with polyethylene glycol (PEG) and exogenous ABA added were used to analyze TaPYR10 expression patterns in response to drought and ABA signals. The PEG concentrations used included 0, 1,5, 10 and 15% (w/v), corresponding to osmotic potentials in solution of 0, -0.35, -0.81, -1.08 and-1.27 MPa, respectively. ABA levels in the MS solutions were 0.5, 1.0, 1.5 and 2.0 μmol. In addition, seedlings collected at different time points (0 hours before treatment and 1, 3, 9 and 27 hours after treatment) under drought (10% peg) and ABA (1 μmol) treatments were used to characterize TaPYR the temporal expression pattern of 10 in response to the above 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, tubulin (Tatubulin) and actin (Taactin) in wheat, were used as internal controls to normalize the target transcripts.

(5) Glucuronidase (GUS) assay

GUS histochemical staining and GUS activity, predicted by the plant cis-acting regulatory element database (PLANTCARE, http:// bioinformation. Psb. Ugent. Be/webtools/plantcare/html /), driven by various cis-acting regulatory elements located in the TaPYR promoter, was measured in transgenic wheat lines carrying truncated promoter fragments. To this end, full length promoter (1764 bp) and a set of promoter regions TaPYR10, 454bp, 745bp, 1378bp and 1666bp, containing various cis-acting regulatory elements (i.e. DRE and MYB, MYC recognition sites) that affect gene transcription and osmotic stress response were amplified from wheat genomic DNA (Dan Mai bp) using gene-specific primers (table 2) (table 3). Transgenic wheat (Dan Mai) lines carrying these cassettes, i.e., taPYR promoter fragment and reporter gene (β -glucuronidase, GUS), were obtained using agrobacterium-mediated transformation methods (Kumar et al, 2019). After 6 hours of drought treatment (10% PEG), representative leaves of the T2 transgenic line were subjected to GUS histochemical staining and activity assessment (Houde et al 2020).

TABLE 3TaPYR drought-related cis-acting element and stress response-related Gene information Table in promoter

(6) TaPYR10 expression profiling of downstream partners

The expression analysis of the TaSLAC family members regulating stomatal movement, the family members involved in proline biosynthesis, the SOD family affecting superoxide dismutase activity, the CAT family affecting catalase activity, and the PIN-FORMED (PIN) family genes controlling auxin transport and root structural behavior in the drought stress TaNF-YC1 transgenic lines was performed. The SLAC1 genes analyzed included TaSLAC-1 to TaSLAC-1, the proline biosynthesis genes included TaP CS1, taP CS2, taP CR1, taProDH1 and TaP CDH1, the SOD genes included TaSOD1 to TaSOD6, the CAT genes included TaCAT1 to TaCAT6, and the PIN genes included TaPIN1 to TaPIN6. Root tissue of trefoil seedlings cultured in modified MS solution (10% peg) was evaluated for target expression levels after 27 hours of treatment. Transcripts of the above genes were detected using gene specific primers (Table 2) as in qRT-PCR procedures performed on TaPYR a 10 under drought stress. Expression patterns of TaSLAC1-3, taP5CR1, taSOD4, taCAT2, taPIN, and TaPYR10 of 45 test wheat varieties (Table 1) were evaluated in the middle of the grouting period under field drought conditions (a water potential of the plough layer soil of-1.05 mPa). Wherein, the transgene and wild plants are planted in the field cells (length 1m, width 0.3 m) and the wheat varieties are planted in the cells (length 8m, width 4 m) and repeated three times. Drought stress is established by conventional deficit irrigation management (irrigation prior to sowing and during the jointing period). Representative flag leaves of the cultivars were collected using gene specific primers (table 2) and target transcripts were evaluated based on qRT-PCR.

(7) Yeast two-hybrid experiments

The partners of the TaPYR participating ABA signaling module, namely PP2C protein, snRK2 kinase and NF-Y transcription factors, were determined using a yeast two-hybrid experiment. PP2C family members from which experiments were conducted included TaPP C2C 3, taPP C5, taPP C7, taPP C10, and TaPP C30, snRK2 family members included TaSnRK2.5 to TaSnRK2.10, and NF-Y family members included TaNF-YC1, taNF-YC2, taNF-YC3, taNF-YC5, and TaNF-YC8 (Table 2). For this purpose, the ORF of TaPYR was amplified using RT-PCR and integrated into the expression vector pGADT7 to produce the cassette pGADT7-TaPYR10 (bait). Meanwhile, the ORF of the PP2C gene was amplified and inserted into the expression vector pGBKT7 as a prey, respectively. Yeast hosts (strain AH 109) containing baits (TaPYR) and prey (each TaPP C protein) were cultured on selective solid medium supplemented with exogenous ABA (1. Mu. Mol) to initiate protein-protein interactions. Using a similar strategy, the ORF of the PP2C member that interacted with TaPYR10 was amplified by RT-PCR as a bait, while the ORF of the SnRK2 member was amplified and inserted into the expression vector pGBKT7, respectively, as a prey. Similarly, the SnRK2 member interacting with the PP2C protein described above was amplified and inserted into the vector pGADT7 as a bait, while the ORF of NF-YC member was amplified and inserted into the expression vector pGBKT7, respectively, as a prey. Positive transformants containing baits and prey were grown on selection medium SD/-Leu/-Trp lacking specific amino acids for 3 days at 30 ℃. Fragments of TaPYR [ i.e., taPYR-N1 (amino acids 1 to 70), taPYR-10-I1 (amino acids 71 to 140) and TaPYR-10-C1 (amino acids 141 to 211) ], taPP-2C 30[ TaPP C30-N1 (amino acids 1 to 73), taPP-2C 30-I1 (amino acids 74 to 317) and TaPP-2C 30-C1 (amino acids 318 to 406) ], taSnRK2.10[ TaSnRK2.10-N1 (amino acids 1 to 90), taSnRK2.10-I1 (amino acids 91 to 260) and TaSnRK2.10-C1 (amino acids 261 to 394) ] and TaNF-YC1[ TaNF-YC1-N1 (amino acids 1 to 80) ] and TaNF-YC1-C1 (amino acids 81 to 255) ] were amplified by RT-PCR using gene specific primers (Table 2). They were then used to construct baits or prey and subjected to a yeast two-hybrid experiment similar to that described above.

(8) Bimolecular fluorescence complementation (BiFC) experiment

A BiFC experiment was performed to verify TaPYR in vivo protein interactions between 10 and its downstream partners. For this purpose, the ORFs of TaPYR, taPP C30, taSnRK2.10 and TaNF-YC1 were amplified using RT-PCR and gene-specific primers (Table 2) and integrated in-frame with the fragment of the reporter gene YFP (yellow fluorescent protein gene) at the C-terminus (pSPYCE (M) -TaPYR and pSPYCE (M) -TaSnRK2.10) or at the N-terminus (pSPYNE (R) -TaPP C30 and pSPYNE (R) -TaNF-YC 1) according to the methods described previously (Shen et al, 2011). The combination of expression cassettes, i.e. pSPYC (M) -TaPYR10 and pSPYNE (R) -TaPP C30, pSPYNE (R) -TaPP C30 and pSPYC (M) -TaSnRK2.10, pSPYC (M) -TaSnRK2.10 and pSPYNE (R) -TaNF-YC1, was used to genetically co-transform Agrobacterium (strain EHA 105) and further transiently transform young leaves of Nicotiana benthamiana (Kumar et al, 2019). After 2 days of infiltration, YFP signals in transgenic leaves were detected using a fluorescence microscope (Olympus FV10-ASW, japan) according to the manufacturer's instructions. While detecting YFP signal, nuclear target was identified using nuclear marker DAPI (Solarbio, beijing, china) according to the previously described method (Sun et al, 2013). The same TaPYR, taPP2C30, taSnRK2.10 and TaNF-YC1 fragments as in the yeast two-hybrid experiment described above were amplified and a BiFC experiment was performed using gene specific primers (Table 2). YFP signals from transformed young leaves of nicotiana benthamiana were detected in a similar manner as described above.

(9) Co-immunoprecipitation (Co-IP) experiments

The ORFs of TaPYR and its downstream partners TaPP C30, taSnRK2.10 and TaNF-YC1 were amplified using RT-PCR and gene-specific primers (Table 2) and cDNA sequences encoding different fragments of the signal members described above (TaPYR I1, taPP C30I1, taSnRK2.10I1 and TaNF-YC1N 1). They were then used to construct expression cassettes, namely MET-TaPYR10/TaPYR I1-GFP, MET-TaPP C30/TaPP C30I1-GFP, MET-TaSnRK2.10/TaSnRK2.10I1-GFP and MET-TaNF-YC1/TaNF-YC1N1-GFP, and to transform Agrobacterium strain EHA105. Positive transformants were used for transient transformation of Nicotiana benthamiana leaves (Zhao et al 2022). Briefly, about 0.3g of leaves were collected after 24 hours of infiltration and ground to a powder using 1mL of buffer A (50 mMhEPES-NaOhph7.5, 150mM NaCl, 5% glycerol, 0.5% Triton X-100, 0.1% (v/v) 3-mercapto-1, 2-propanediol, one Complete Mini protease inhibitor cocktail (Shangon, shanghai, china)) per 10 mL. The resulting solution was centrifuged at 1000g twice at 4℃for 10 minutes to obtain a protein component. They were further mixed with an equal volume of 2 Xdodecyl sodium sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer [ (Laemmli sample buffer (BIO-RAD, calif., U.S.A.) 950. Mu.l/mL, 3-mercapto-1, 2-propanediol 50. Mu.l/mL) ] and denatured at 95℃for 5 min, then stored at 30 ℃. Proteins were incubated with GFP-trap agarose beads (Chromotek, planegg, germany) at 4℃for protein binding. After washing the beads 4-6 times with buffer A, the proteins were eluted with 1 XSDS-PAGE sample buffer (50% 2 XSDS-PAGE sample buffer, 50%1 XPBS). Co-immunoprecipitation experiments were performed using a 12.5% SDS-PAGE gel (e-PAGEL) (ATTO, tokyo, japan).

(10) In vitro pulldown experiments

In vitro pulldown experiments were performed to verify the protein interactions shown in the Co-IP experiments described above. The ORFs of TaPYR, taPP C2C 30, taSnRK2.10 and TaNF-YC1 were amplified by RT-PCR using gene specific primers (Table 2) and cDNA sequences encoding the different fragments of the signal members described above, and cloned into the glutathione S-transferase (GST) tagged protein expression vector pGEX-4T-1, respectively. Simultaneously, the ORF of the TaPYR downstream partner and cDNA sequences encoding the different protein fragments (TaPP C30I1, taSnRK2.10I1 and TaNF-YC1N 1) were amplified and inserted into the expression vector pMAL-C2X with a Maltose Binding Protein (MBP) tag, respectively. The plasmid was routinely transformed into E.coli BL21 (DE 3) strain for protein expression. Positive transformants were collected at 4℃and then resuspended in GST pulldown buffer (20 mM Tris-HCl, ph8.0, 200mM NaCl, 1mM MgCl2 and 0.5% LgepalCA-630) and sonicated in an ice bath. The resulting supernatants were separated and subjected to SDS-PAGE to detect protein components. An equal amount of TaPYR-GST and its downstream proteins were incubated in 500. Mu.l GST pulldown buffer at 4℃for 6 hours, then 100. Mu.l GST beads (GEHEALTHCARE, new Jersey, USA) were added to the mixture and incubated at 4℃for 2 hours. The pulled down proteins were detected using anti-MBP antibodies (NEW ENGLAND Biolabs, los Angeles, USA) and anti-GST antibodies (Abcam, cambridge, UK) according to the manufacturer's instructions.

(11) Construction of transgenic wheat lines

Transgenic wheat lines TaPYR and its downstream partners TaPP C30, taSnRK2.10, taNF-YC1 and TaSLAC-3, taP CR1, taSOD4, taCAT2 and TaPIN6 regulated by TaNF-YC1 were constructed to investigate their role in mediating drought responses. Briefly, the ORFs of the above genes were amplified in either forward or reverse direction by RT-PCR using gene specific primers (Table 2). The amplified products were integrated into the binary vector pCAMBIA3301 at the NcoI/BastEII locus, respectively, under the control of the CaMV35S promoter. The resulting expression cassette was used to genetically transform Agrobacterium (strain EHA 105) which was then further transformed into common wheat (variety Dan Mai) (Kumar et al, 2019). The target transcripts in the transgenic lines were assessed by qRT-PCR using gene specific primers (table 2). Representative strains of each gene over-expressed and knockdown expressed, as well as Wild Type (WT), were selected at T3 generation, cultured under normal conditions, and subjected to drought or ABA treatment.

(12) Establishment of drought and ABA treatments

Drought and ABA treatments were established for the TaPYR and its downstream partners transgenic lines to characterize the function of the genes in mediating plant stress responses. For drought treatment, transgenic lines and wild-type (WT) are grown in growth chambers or under field conditions. For growth chamber cultures, transgenic and WT seedlings were planted in plastic pots containing a soil mixture (half vermiculite and half fertile soil) and watered daily to maintain 70-75% relative soil humidity. They were divided into two groups during the trefoil period, one group grown under normal watering conditions and the other group drought treated by limiting water supply to maintain 55-60% relative soil humidity (using soil water potential apparatus TRS-IIN, zhejiang, china detection). For field planting, transgenic and WT plants were routinely planted in field plots (1 m long by 0.3m wide) and repeated three times. Two irrigations (pre-sowing and jointing stage) were carried out, which was considered to be the conventional deficit irrigation management for local winter wheat (soil water potential of the plough layer in the grouting stage is-1.05 to-1.22 mPa). ABA treatment was established by external spraying of seedlings or plants with 1 μmolABA two weeks prior to the experiment for determination of growth, physiological and biochemical indicators.

(13) Assessment of growth traits and physiological indicators

After drought treatment, transgenic and WT plants were assessed for growth traits and drought-related physiological indices. The assessed growth traits include phenotype and plant biomass. Wherein the phenotype was recorded by taking photographs using a digital camera, plant biomass was obtained from a representative five seedlings (growth chamber culture) or twenty plants (field planting) after drying in an oven, using conventional methods. At maturity, the seeds were phenotypically recorded and the grain weight was assessed after the seeds were air-dried, and cell yield was measured based on the air-dried grain weight of 0.5m ² plants. The physiological indicators evaluated include indicators related to osmotic stress response, such as osmotic regulating substance content, stomatal Closure Rate (SCR), in vitro leaf loss rate (WLR), active oxygen related parameters, using upper expanded leaves as samples. Wherein the content of the osmoregulating substance proline and soluble sugars is assessed (Zhao et al 2024). The pore closure rate (SCR) value was determined from the ratio of pore width to 0 hour observed at different drought time points [ 0.25, 0.5 and 1 hour during 1 hour drought treatment (10% peg) ] (Zhao et al, 2022). The in vitro leaf loss rate (WLR) values were determined from the decrease in fresh weight recorded relative to 0 hours at various time points (0.5, 1 and 3 hours) during drought treatment (starting with leaf placement on a clean bench). Reactive oxygen related indicators such as superoxide dismutase (SOD), catalase (CAT) and Peroxidase (POD) activities, malondialdehyde (MDA) content and accumulation of reactive oxygen (i.e., superoxide anion and H ₂O₂) were evaluated as follows, enzyme activity and MDA content (Huang et al, 2010) were determined, superoxide anion and H ₂O₂ amounts were evaluated using a histochemical staining method, i.e., nitroblue tetrazolium (NBT) staining for superoxide anion, 3' -Diaminobenzidine (DAB) staining for H2O2 (Zhao et al, 2024).

(14) Transcriptional activation assay

Transcriptional activation experiments were performed on five differentially expressed genes, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN, in TaNF-YC1 transgenic lines of TaNF-YC1 in drought stress in the Nicotiana benthamiana expression system. For this purpose, the ORF of TaNF-YC1 was amplified using RT-PCR and gene-specific primers (Table 2) and integrated into the pGreenII-SK vector to produce the cassette CaMV35 Spro:TaNF-YC 1. Meanwhile, the promoter regions (2 kb long) of TaSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 were amplified using wheat (Dan Mai) genomic DNA as a template and gene-specific primers (Table 2) and integrated into pGreenII0800-LUC vectors to generate reporter constructs TaSLAC1-3pro:: luc, taP5CR2:: luc, taSOD4:: luc, taCAT2:: luc and TaPIN pro::: luc, respectively. The recombinant binary cassette was co-transformed onto Agrobacterium strain EHA105 in a 1:1 (v: v) ratio, combining CaMV35 Spro:TaNF-YC 1 and TaSLAC-3 pro:LUC, caMV35 Spro:TaNF-YC 1 and TaP CR 1:LUC, caMV35 Spro:TaNF-YC 1 and TaSOD4 pro:LUC, caMV35 Spro:TaNF-YC 1 and TaCAT pro:LUC, caMV35 Spro:TaNF-YC 1 and TaPIN pro:LUC. Positive transformants were further used for transient transformation of B.benthamiana epidermal cells (Wang et al 2020). After 48 hours of transformation and 100mM fluorescein sprayed, the reporter fluorescein signal in transiently transformed cells was evaluated using a charge coupled device imaging device (NightOWLII LB983 in combination with Indigo software) according to the manufacturer's instructions.

(15) Yeast Single hybridization experiment

Yeast single hybridization assays were performed following the previous methods (Zhang et al, 2024) to verify transcriptional activation of TaNF-YC1 on its target gene, taSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN 6. For this purpose, the ORF of TaPYR was amplified by RT-PCR using gene specific primers (Table 2) and integrated into pGADT7 vector (Clontech) at EcoRI and KpnI sites to produce the fusion protein pGADT7-TaPYR10 (as a prey). Meanwhile, the promoter regions of their target genes were inserted into XhoI and KpnI sites of pHIS2 reporter vector (Clontech) to produce fusion proteins pHIS2-TaSLAC1-3, pHIS2-TaP CR1, pHIS2-TaSOD4, pHIS2-TaCAT2 and pHIS2-TaPIN6, respectively (as baits). The combination consisting of prey and each decoy plasmid was co-transformed into a yeast strain (EGY 48). Positive transformants were identified after 3 days of culture on solid selective growth medium lacking SD-Trp/-Ura.

(16) Statistical analysis

The average of gene transcripts, growth traits, plant biomass and osmotic stress related physiological indicators was from triplicate results. Statistical analysis system software (SAS Corporation, cary, NC, USA) was used to determine standard error of the mean and to evaluate statistical significance analysis of the traits. Regression analysis results between plant biomass and TaPYR10, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 expression levels in the variety population under field drought conditions were assessed using analysis of variance (ANOVA). Statistical significance analysis of transgenic lines and WT on plant morphological traits and physiological indexes was determined using Student's t-test (threshold P≤0.05). A heat map showing TaPYR and stress responsive gene expression levels and wheat variety yield in field experiments was drawn using software called TBtools.

Example 2, results

(1) Molecular characterization of TaPYR10

TaPYR10 cDNA length 633bp, encoding a polypeptide consisting of 211 amino acids, molecular weight 22.62kDa, isoelectric point (pI) 4.97.

At the amino acid level TaPYR has a high similarity to its counterparts from wheat (t.dicoccoids), rye (a.tauschii), seashore paspalum (p.haliii), millet (p.milicaceum), corn (z.mays), brachypodium (b.discochyon), sorghum (s.bicolor) and brachypodium (o.bromophytha), all of which contain a conserved pyr_pyl_ PCAR domain involving a composition of nine leaf units (fig. 1A).

At the nucleic acid level TaPYR has a high degree of identity with homologous genes in different plant species, with the highest similarity with genes from seashore paspalum (p. Hali), switchgrass (p. Virgatum), green bristlegrass (s. Virdis), japanese rice (z. Japonica), maize (z. Mays), short oat (o.brachytrium), barley (h. Vulgare), festival wheat (a. Tausch), and two wheat (t. Dicoccides) (fig. 2).

These results indicate that TaPYR has a similar evolutionary pathway to its plant counterparts. Based on three-dimensional structural (3-D) analysis, taPYR was found to contain one hexalobal β -propeller domain and three α -helices, which together form nine structural sites that constitute the pyr_pyl_ PCAR domain. Furthermore TaPYR comprises an A8S type binding site for abscisic acid. These binding sites are involved in binding ABA and interacting with downstream proteins (fig. 1B).

In model plants, nicotiana benthamiana (n.benthamiana) and wheat protoplasts, transiently transformed cells expressing TaPYR-GFP fusion proteins showed GFP signal localization to the nucleus (fig. 1C), suggesting that TaPYR protein localizes to the nucleus and is involved in biological function after sorting through the Endoplasmic Reticulum (ER).

(2) TaPYR10 expression sensitivity of 10 to drought signals

Analysis of TaPYR transcripts revealed their expression changes in response to drought and ABA signals. Under conditions of increased drought, taPYR expression levels in both roots and leaves were significantly up-regulated, reaching the highest level at 15% peg treatment (fig. 3A). In addition, taPYR transcripts in both tissues gradually increased during 27 hours of drought treatment and recovered to normal levels after 27 hours of recovery conditions (fig. 3A). A similar TaPYR expression pattern was also observed in exogenous ABA treated plant tissues, transcripts of the wheat PYR gene proved to be induced by ABA signal, in ABA concentration and time dependence (FIG. 3B). These findings indicate that TaPYR a 10 is highly sensitive to drought and ABA signals at the transcriptional level. On-line predictive analysis showed that TaPYR promoter contained a set of potential cis-acting regulatory elements associated with gene transcription under osmotic stress, including conserved motifs TATA box and CAAT box that regulate gene transcription, elements DRE that respond to osmotic stress, and binding sites for transcription factors MYB and MYC that mediate gene transcription under stress (fig. 3C-D). These cis-regulatory elements are thought to play an important role in regulating TaPYR transcription under drought conditions. GUS staining and GUS activity enhancement in leaves transformed with full length promoter (D5,1764 bp) and truncated TaPYR promoter fragments D1 (454 bp), D2 (745 bp), D3 (1378 bp) and D4 (1666 bp) under drought conditions compared to control (non-stress conditions) (FIG. 3D-F). Furthermore, GUS staining and activity in leaves gradually increased with extension of the promoter fragment (FIG. 3D-F). These results indicate that cis-element DRE and MYB and MYC transcription factor binding sites synergistically regulate TaPYR transcription in response to drought and ABA signals.

(3) TaPYR10 and TaPP C30, taSnRK2.10 and TaNF-YC1 form an ABA core signal module

On-line predictive analysis showed that TaPYR protein could interact with PP2C member TaPP C30, which could interact with SnRK2 member tasnrk 2.10. Similarly, the above-mentioned SnRK2 member interacts with the NF-YC member TaNF-YC1 (FIG. 4A). Based on the predicted results, experimental analysis was performed on interactions between TaPYR and its putative downstream partner. The role of TaPYR in establishing ABA signaling modules was studied by yeast two-hybrid, two-molecule fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and in vitro pulldown experiments. In a yeast two-hybrid experiment, taPYR protein (bait) expressed in yeast cells specifically interacted with PP2C member TaPP C30 (prey) expressed in yeast host (fig. 4B). Likewise, the TaPP C30 protein was found to interact specifically with TaSnRK2.10, and the latter interacted with TaNF-YC1 in the same experimental system (FIGS. 4C-D). The protein-protein interactions identified from the yeast two-hybrid experiments were further confirmed by BiFC, and clear YFP signals were detected in Nicotiana benthamiana epidermal cells co-transformed with TaPYR-nYFP and TaPP C30-cYFP, taPP2C30-cYFP and TaSnRK2.10-nYFP, and TaSnRK2.10-nYFP and TaNF-YC1-cYFP (FIG. 4E). Co-IP and in vitro pulldown analysis of the above ABA signal components validated protein interaction results obtained from yeast two-hybrid and BiFC experiments. Co-IP analysis showed, among other things, that TaPYR10 was able to Co-immunoprecipitate with TaPP C30, taPP C30 was able to Co-immunoprecipitate with TaSnRK2.10, and TaSnRK2.10 was able to Co-immunoprecipitate with TaNF-YC1 (FIGS. 4F-H). In vitro pulldown analysis confirmed the protein interactions between the ABA signaling partners described above (fig. 4I-K). These findings indicate that TaPYR forms with its downstream partners TaPP C30, taSnRK2.10 and TaNF-YC1 an ABA core signaling module TaPYR/TaPP C30/TaSnRK2.10/TaNF-YC1 that plays a critical role in transducing stress-induced ABA signals and affecting drought response.

(4) TaPYR10 and protein fragments interacting with downstream partners thereof

To investigate the different regions involved in protein-protein interactions between TaPYR, taPP2C30, tasnrk2.10 and TaNF-YC1, their N-and C-terminal fragments and intermediate fragments of TaPYR and TaPP C30 (fig. 5A) were expressed and analyzed using a protein interaction experiment. In a yeast two-hybrid experiment, positive yeast transformants were identified in cells co-expressing TaPYR intermediate fragment (TaPYR-I1, amino acids 71 to 140, containing the CL3-CL5 loop and the PYR/PYL/PCAR conserved domain) and TaPP C30 intermediate fragment (TaPP C30-I1, amino acids 74 to 317, containing the PP2C conserved activating domain) (FIG. 5B). Likewise, yeast host cells co-expressing TaPP C30-I1 and the intermediate fragment of TaSnRK2.10 (TaSnRK2.10-I1, amino acids 91 to 260, containing conserved serine/threonine kinase activation sites) and yeast host cells co-expressing TaSnRK2.10-I1 and the N-terminus of TaNF-YC1 (TaNF-YC 1-N1, amino acids 1 to 80, containing nuclear localization signals) showed complementary growth capacity on selection medium SD/-Leu/-Trp lacking amino acids (FIG. 5B). In the yeast two-hybrid experiments, no interactions between TaPYR and other PP2C proteins, taPP C30 and other SnRK2 members, and tasnrk2.10 and other NF-YC proteins were detected other than the downstream partner of TaPYR, described previously (fig. 6). The BiFC experiment confirmed the interaction between these fragments in TaPYR10 and its downstream signal partners. The reporter gene YFP signal was detected with high sensitivity in N.benthamiana epidermal cells co-transformed with TaPYR-I1-nYFP/TaPP 2C30-I1-cYFP, taPP2C30-I1-cYFP/TaSnRK2.10-I1-nYFP and TaSnRK2.10-I1-nYFP/TaNF-YC1-N1-cYFP (FIG. 5C). Further Co-IP and in vitro pulldown analysis confirm the above interaction procedure obtained from yeast two-hybrid and BiFC experiments. Both experiments showed that TaPYR I1 and TaPP C30I1 co-immunoprecipitated, taPP C30I1 co-immunoprecipitated with TaSnRK2.10I1, and TaSnRK2.10I1 co-immunoprecipitated with TaNF-YC1N1 (FIG. 5D-I). These findings indicate that the distinct protein regions in TaPYR and its downstream partners contribute to protein-protein interactions between ABA signaling pathway members.

(5) TaPYR transgenic lines of TaPYR and its downstream partner genes alter plant drought response

A transgenic wheat line of TaPYR and its downstream partner genes was constructed to characterize the function of the genes in mediating drought responses in plants. Under normal growth conditions, lines TaPYR10 (Sen 1, sen2, anti 1 and Anti 2), taPP C30 (Sen 2, sen 3, anti 1 and Anti 2), tasnrk2.10 (Sen 1, sen2, anti 2 and Anti 3) and TaNF-YC1 (Sen 2, sen 3, anti 2 and Anti 3) had significant changes in target gene expression (fig. 7), but were similar to Wild Type (WT) plants in terms of growth traits, i.e. phenotype, biomass, leaf area, grain weight and yield (fig. 8A-T, fig. 9). Under drought conditions, the growth characteristics of all transgenic lines changed. Strains overexpressing TaPYR (Sen 1 and Sen 2), taSnRK2.10 (Sen 1 and Sen 2) and TaNF-YC1 (Sen 2 and Sen 3) and TaPP C30 knockdown (Anti 1 and Anti 2) showed increased phenotype (FIGS. 8A-D), increased biomass (FIGS. 8E-H), increased leaf area (FIGS. 8I-L), increased grain weight (FIGS. 8M-P) and increased yield (FIGS. 8Q-T) compared to wild-type (WT) plants. In contrast, taPYR (Anti 1 and Anti 2), the lines knocked down by TaSnRK2.10 (Anti 2 and Anti 3) and TaNF-YC1 (Anti 2 and Anti 3) and the lines overexpressing TaPP C30 (Sen 2 and Sen 3) had a deteriorated phenotype (FIGS. 8A-D), and plant biomass, leaf area, grain weight and yield were reduced relative to WT plants (FIGS. 8E-T). The growth behavior of the transgenic lines described above under exogenous ABA treatment was similar to that described above under drought conditions (fig. 10). Alterations in the growth traits of these transgenic lines indicate that TaPYR and its signal pathway genes play a key role in regulating plant drought and ABA responses.

(6) TaPYR osmotic stress related physiological properties of TaPYR and its downstream partner transgenic lines

Physiological characteristics and indices related to osmotic stress response in TaPYR, taPP, 2C30, tasnrk2.10 and TaNF-YC1 lines were evaluated, including Stomatal Closure Rate (SCR), in vitro leaf loss rate (WLR), content of the osmoregulating substance proline and soluble sugars, photosynthetic parameters, reactive oxygen species related indices and root morphology features. Within 1 hour of drought treatment, the lines with TaPP C30 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) over-expressed increased stomatal closure compared to WT (FIGS. 11A-E). During 2 hours of leaf dehydration, the in vitro leaf loss rates of these lines were on a decreasing trend relative to WT plants (fig. 11F-I). These lines also showed increased levels of proline (FIG. 11J-M) and soluble sugars (FIG. 11N-Q), increased photosynthetic capacity (net photosynthetic rate Pn, gas pore conductance Gs, and actual quantum efficiency of photosystem II. Phi. PSII were increased, non-photochemical quenching coefficient NPQ was decreased) (FIG. 12) and improved reactive oxygen species-related index, i.e., decreased levels of superoxide anions and H ₂O₂ (FIGS. 13A-H, FIG. 14A-H), superoxide dismutase (SOD), catalase (CAT), and Peroxidase (POD) activities (FIGS. 13I-T), decreased Malondialdehyde (MDA) accumulation (FIGS. 15A-D). The Root Structure (RSA) characteristics of these transgenic lines, i.e., root morphology (FIG. 13U-X), root biomass (FIG. 15E-H), and root volume (FIG. 15I-L), are consistent with their role in mediating drought responses in plants. In contrast, the TaPP C30 overexpressing (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 exhibited opposite effects under drought treatment, i.e., reduced stomatal closure rate, increased in vitro leaf water loss rate, reduced proline and soluble sugar content, reduced photosynthetic capacity, worsening of reactive oxygen steady state related indicators (FIGS. 11A-Q, 13A-T, 15A-D) and reduced root structure related characteristics (FIGS. 13U-X, 15E-L). Overall, taPYR signaling modules play an important role in mediating plant stress response-related physiological processes.

(7) Expression of TaNF-YC1 transcriptional activation osmotic stress response gene

Expression analysis was performed on a panel of genes including SLAC1 family genes (TaSLAC 1-1 to TaSLAC 1-6) regulating stomatal movement, genes associated with proline accumulation (TaP CS1, taP CS2, taP CR1, taProDH1, taP CDH 1), superoxide dismutase (SOD) family (taso 1 to taso 6) and Catalase (CAT) family (TaCAT to TaCAT) genes mediating antioxidant enzyme activity, and PIN-FORMED (PIN) family genes (TaPIN to TaPIN) regulating root structure establishment, which were analyzed in the TaNF-YC1 transgenic lines after drought and exogenous ABA treatment. Among the genes examined, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 were altered in expression levels in the TaNF-YC1 strain relative to the WT strain (higher expression levels in Sen 2 and Sen 3 and lower expression levels in Anti 2 and Anti 3) (FIGS. 16A-E). In contrast, in drought-treated TaNF-YC1 transgenic lines, the transcripts of the other detected genes were unchanged compared to WT (FIGS. 16A-E). These results indicate that differentially expressed genes TaSLAC1-3, taP5CR1, taSOD4, taCAT2, and TaPIN6 are affected by TaNF-YC1 at the transcriptional level. Yeast single hybridization experiments were performed to determine the protein/DNA interactions between TaNF-YC1 and the differentially expressed gene promoter. FIG. 16F is a schematic diagram showing the distribution of cis-elements in stress response genes in response to drought (MYB) and ABA (ABRE). As expected, hosts co-transformed with pGADT7-TaNF-YC1 and pHIS2-TaSLAC1-3Pro, pGADT7-TaNF-YC1 and pHIS2-TaP CR1Pro, pGADT7-TaNF-YC1 and pHIS2-TaSOD4Pro, pGADT7-TaNF-YC1 and pHIS2-TaCAT2Pro and pGADT7-TaNF-YC1 and pHIS2-TaPIN4Pro were grown normally on triple-deficient medium (SD/-Leu-Trp-His) plates containing 45mmol L ^-1 -AT (FIG. 16G), indicating that TaNF-YC1 interacts with the promoter of the stress response gene. A transcriptional activation experiment was performed in Nicotiana benthamiana to verify the regulation of the above stress defense gene by TaNF-YC1, and a strong reporter LUC signal was detected in tobacco epidermal cells transiently co-transformed in combination with the following plasmids, effector cassette CaMV35SPro:: taNF-YC1 and reporter cassettes TaSLAC-3 Pro:: LUC (FIG. 16H, M), effector cassette CaMV35SPro:: taNF-YC1 and reporter cassette TaP CR1 Pro::: LUC (FIG. 16I, N), effector cassette CaMV35SPro: taNF-YC1 and reporter cassette TaSOD4Pro:: LUC (FIG. 16J, O), effect boxes CaMV35SPro:: taNF-YC1 and report box TaCAT Pro:: LUC (FIG. 16K, P), and effect boxes CaMV35SPro:: taNF-YC1 and report box TaPIN6Pro:: LUC (FIG. 16L, Q). together, these results demonstrate that TaSLAC1-3, taP5CR1, taSOD4, taCAT2, and TaPIN6 transcription is regulated by TaNF-YC1 and affects osmotic stress related physiological processes under drought conditions.

(8) Adaptation of differential osmotic stress response gene forward regulation to drought stress

Transgenic analyses were performed on differentially expressed stress response genes TaSLAC1-3, taP5CR1, taSOD4, taCAT2, and TaPIN6 to characterize their role in mediating drought responses. Under drought treatment, the TaSLAC1-3 knockdown lines (AntiSLAC 1-3A and AntiSLAC 1-3B) grew slowly, biomass decreased, proline content decreased (FIG. 17D-F), taSOD4 knockdown (AntiSOD-2 and AntiSOD 4-3) resulted in altered phenotypes, decreased SOD activity, increased accumulation of superoxide anions (FIG. 17G-I, FIG. 18A), the TaCAT2 knockdown lines (AntiCAT 2-2 and AntiCAT 1-3) had a poorer phenotype, decreased CAT activity, increased accumulation of H ₂O₂ (FIG. 17J-L, FIG. 18B), the TaPIN6 knockdown lines (AntiPIN 6-1 and AntiPIN 6-2) had significantly reduced root growth, decreased root biomass, and reduced root volume (FIG. 17M-O) compared to the WT plants. Without drought treatment, these transgenic lines were not significantly different from WT plants in plant phenotype and root growth. These results demonstrate the positive effect of osmotic stress-related genes on drought adaptation at the TaPYR module member, taNF-YC1, transcript level.

(9) TaPYR10 in wheat variety under field drought condition and transcript abundance and yield of downstream stress defense genes thereof are highly related

Expression levels of TaPYR and stress-responsive genes (TaSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN 6) were tested in a group of 45 wheat varieties with different drought tolerance in plant yield expression. Under field drought conditions, transcripts of these genes showed significant differences in upper leaves in mid-grout (fig. 19A). Also, under drought conditions in the field, there was a large difference in the yield of the wheat varieties examined at maturity (fig. 19B). Regression analysis was performed to characterize the relationship between gene transcripts and yield in wheat varieties under drought stress. The results showed that yields were significantly positively correlated with the expression levels of TaPYR and four stress-responsive genes (fig. 19C-H). These findings indicate that TaPYR and different stress defense genes act synergistically at the transcriptional level, regulating plant drought response and contributing to plant drought adaptation.

(10) TaPYR10 haplotype variant behavior of 10

To identify the sequence variation of TaPYR a Single Nucleotide Polymorphism (SNP) site in the ORF and a SNP site in the promoter region were detected by sequencing the Open Reading Frame (ORF) and flanking regions (i.e., 2kb upstream of the start codon) of 45 wheat varieties. The 4 SNPs in the ORF of TaPYR and the 5 SNPs in the promoter region form two major haplotypes, taPYR-Hap 1 and TaPYR-Hap 2 (FIG. 20A). Based on the above results, a competitive allele-specific PCR (KASP) marker, designated TaPYR-KASPA 523T, was developed and used to genotype 45 elite wheat varieties tested (FIG. 20B). Expression levels of TaPYR and stress response genes (TaSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN 6) were characterized. Thermogram analysis showed that wheat cultivars classified as TaPYR-Hap 1 had higher expression levels than cultivars classified as TaPYR-Hap 2 (fig. 20C), resulting in plants with higher proline content, plant biomass and yield (fig. 20D-F). Together, these results indicate TaPYR-Hap 1 was positively selected in modern wheat breeding programs.

Example 3 conclusion

(1) TaPYR 10A response to drought signals correlates with specific cis-elements in the promoter

The expression of ABA signaling genes is tightly regulated at the transcriptional level. For example, transcripts of PYR family genes may change in response to drought, salt and ABA stresses due to increased ABA levels in plant cells (Antoni et al 2012; li et al 2018). Drought and exogenous ABA treatment can 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 the heterotrimer formed by nuclear factor YC subunit (NF-YC) family transcription factor members GmNF-YC14, gmNF-YA16 and GmNF-YB2 in soybean activates GmPYR 1-mediated abscisic acid (ABA) signaling pathways, thereby regulating soybean stress tolerance (Yu et al, 2021). In this study, our analysis of TaPYR expression showed that it is responsive to drought and ABA signals, and its expression level in roots and leaves depends on the extent and duration of stress. These findings indicate that TaPYR is precisely regulated at the transcriptional level by drought and ABA signals.

Transcriptional changes in drought-responsive genes depend on cis-acting regulatory elements located on their promoters. Elements such as drought and ABA response elements (DREs), recognition sites for transcription factors MYB and MYC 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 the TaPYR promoter that control gene transcription, including TATA and CAAT boxes, as well as elements that elicit gene responses to ABA and drought stress, i.e., recognition sites for DRE and MYB, MYC transcription factors. Further GUS histochemical staining and activity assays verified their function in regulating gene response to drought and ABA signaling by transformation of wheat leaves using a series of truncated promoter fragments driving the expression of the reporter gene GUS. Additional experiments using base mutation methods can help determine the mechanism by which these cis-elements control gene transcription under drought stress.

(2) TaPYR10 and downstream partners TaPP C30, taSnRK2.10 and TaNF-YC1 constitute the ABA core signaling pathway. ABA signaling pathway functional modules involved in mediating ABA and drought responses have been established in plant species (Asad et al, 2019). Accumulation of ABA and formation of the PYR/PYL/RCAR-PP2C complex lead to PP2C inactivation, followed by activation of SnRK2 under stress conditions. Activated SnRK2 promotes transcription of ABA responsive genes by phosphorylating downstream substrates (Ali et al 2020). However, in cereal crops, the downstream partner of the ABA signaling member that interacts with ABA receptors remains 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 wheat ABA signaling pathway involving TaPYR. Our results indicate that TaPYR interacts with its downstream partners, namely TaPP C30, taSnRK2.10 and TaNF-YC 1. These findings indicate the presence of a wheat ABA signaling pathway consisting of these proteins (TaPYR/TaPP 2C30/TaSnRK2.10/TaNF-YC 1). Previous literature reported that ABA receptors can play a role in the nucleus (Cutler et al, 2010), and that most PP2C family gene members were also found to localize to the nucleus. Intracellular localization of the ABA signaling partner contributes to the specificity of PP2C-RCAR pairing and its binding properties to downstream acting SnRK2 (Tischer et al, 2017). In this study, our BiFC experiments revealed that TaPYR10 and its downstream partners interact at the site in the nucleus. This is consistent with the localization reported in some previous studies, suggesting that this may be where they perform biological functions. To date, there has been limited research on the different regions of protein-protein interactions between ABA receptors and their downstream partners. In this study we determined fragments involved in protein interactions between TaPYR protein and its downstream partner. Our results indicate that the middle part of TaPYR10 (amino acids 71 to 140), the middle part of TaPP C30 (amino acids 74 to 317), the middle part of tasnrk2.10 (amino acids 91 to 260) and the N-terminus of TaNF-YC1 (amino acids 1 to 80) contribute to the protein interaction between TaPYR and its downstream partner. Further characterization of the fine localization of the control interactions in these fragments can help understand the molecular processes of protein interactions in the ABA signaling pathway in common wheat.

(3) TaPYR10 and its downstream partners exert a significant influence on plant drought response by modulating stress-related physiological indicators. ABA receptor gene mediated plant drought and ABA responses occur through ABA-dependent signaling pathways (Park et al 2009; gonz lez-Guzm an et al 2014). In view of their significant involvement in regulating stress defense physiological processes, including stomatal movement, osmoregulation substance biosynthesis, cellular reactive oxygen homeostasis and root system structure (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 receptor of tomato is highly expressed in roots and has the ability to enhance drought tolerance in plants (Gonz a lez-Guzm a n et al, 2014). PePYL4 enhances drought tolerance in poplar by regulating water utilization efficiency and active oxygen scavenging (Li et al 2022). These studies have enhanced our understanding of the mechanisms by which the PYR1/PYL/RCAR genes regulate drought adaptation in plants. Similarly, studies of ABA receptor downstream genes have also shown that they play a key role in mediating drought responses. SnRK2 family member tasnrk2.9 confers drought tolerance to transgenic tobacco plants (Feng et al, 2019). The PP2C family member MePP C24 in cassava affects plant drought response through ABA dependent pathways (Zeng et al, 2024). In this study we constructed TaPYR and its downstream partners, namely the TaPP C30, tasnrk2.10 and TaNF-YC1 transgenic lines, including over-and knock-down expression lines, to characterize their function in mediating drought response. Our results demonstrate positive effects of TaPYR, taSnRK2.10 and TaNF-YC1, while demonstrating the negative function of TaPP C30 in regulating plant growth under drought conditions. Drought treatment of TaPYR.sup.10 transgenic lines under field conditions was demonstrated to be positive for this wheat PYR member in mediating plant adaptation to drought stress. These transgenic analysis results show that these genes are valuable for engineering drought tolerant varieties of common wheat. Transgenic analysis of TaPYR, taPP2C30, taSnRK2.10 and TaNF-YC1 in this study demonstrated their role in regulating these physiological processes in regulating drought response.

To characterize the molecular processes of TaPYR modules in regulating osmotic stress-related physiological processes, we analyzed the expression pattern of each gene module to assess its effect on each trait, i.e., slow anion channel 1 (SLAC 1) family genes (TaSLAC-1 through TaSLAC 1-6) regulate stomatal movement by regulating stress-induced anion outflow (Linder and Raschke,1992; schroeder and Keller, 1992), proline accumulation-related genes (TaP CS1, taP5CS2, taP CR 1), TaProDH1, taP5CDH 1) as a limiting factor for proline biosynthesis (Bandurska et al, 2017), antioxidant Enzyme (AE) members such as SOD and CAT family genes affect cellular reactive oxygen homeostasis (Hong et al, 2024), and PIN-FORMED (PIN) family genes control auxin transport and RSA behavior (Zhang et al, 2019; pei et al, 2012; li et al, 2014; zhou and Luo, 2018). In drought-stressed TaNF-YC1 transgenic lines, specific gene family members, including TaSLAC1-3, taP5CR1, taSOD4, taCAT2, and TaPIN, were differentially expressed. Compared to the wild type, these genes had higher transcripts in the transgenic TaNF-YC1 overexpressing lines and lower transcripts in the knockdown lines after drought treatment. These results indicate that these genes have potential roles in TaNF-YC1 regulated drought response. We performed a yeast single hybridization experiment to determine the protein/DNA interactions between TaNF-YC1 and the promoter regions of these stress defense genes to see if these genes are regulated at the transcriptional level by wheat NF-Y transcription factors. Our results indicate that TaNF-YC1 interacts with the promoters of these genes by specifically binding to different promoter regions comprising ABRE or MYB, 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 TaPIN 6) verified that they are all regulated by TaNF-YC 1. In addition, transgenic analysis demonstrated their biological role in regulating antioxidant enzyme activity of stomatal closure (TaSLAC 1-3), proline accumulation (TaP CR 1), SOD (TaSOD 4) and CAT (TaCAT 2), and root morphology (TaPIN). In summary, taPYR module plays a key role in regulating plant drought response by regulating a series of stress response genes regulated by TaNF-YC 1at the transcriptional level. Genetic variation in the expression of # TaPYR and stress response genes contributing to different drought tolerance of wheat varieties is considered as an essential element in enhancing quantitative traits of crops, including yield composition and drought tolerance. Various methods have been employed to expand genetic variation of crop plants, such as the introduction of existing varieties, the development of segregating materials by local or international nursery, crossing and mutation breeding (Thudi et al, 2021). Furthermore, the use of parental lines with different genetic backgrounds, including unrelated and complementary genetic resources with appropriate drought adaptation and yield increasing traits, can help create superior breeding populations (Thudi et al, 2021). In this study, we assessed the expression levels of TaPYR and stress response genes (i.e., taSLAC-3, taP5CR1, taSOD4, taCAT2, and TaPIN 6) in a panel of core varieties consisting of 45 wheat varieties with different plant drought responses. In field experiments under water-saving irrigation management, the expression levels of these genes in flag leaves during the middle of grouting have significant differences among varieties. In addition, plant yield was found to be highly positively correlated with expression levels of TaPYR10, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN 6. Taken together, these findings indicate that genes TaPYR10, taSLAC1-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 play a critical role in regulating plant yield production. Our findings indicate that transcript abundance of the TaPYR-10-composed signaling module can be used as an effective index for assessing drought adaptation of wheat varieties grown under drought conditions. Based on characterization of SNP behavior of TaPYR promoter in wheat variety panels, we revealed base variation of the region flanking MYB recognition site in promoters of different drought tolerant wheat varieties. Two haplotypes, including TaPYR-Hap 1 and TaPYR10-Hap2, are thought to affect plant drought response by affecting transcript abundance of the target gene. Haplotype TaPYR-Hap 1 gives better drought adaptability to wheat varieties, which shows that the haplotype can be a valuable target of common wheat molecular breeding drought-tolerant varieties. Based on our studies, we established a working model of TaPYR a 10 and its downstream partners in mediating plant drought responses (figure 21). In plant tissues, taPYR transcripts were up-regulated under drought and ABA signal stimulation. The induced TaPYR protein, upon binding to an ABA molecule, interacts with PP2C family member TaPP C30 through a protein-protein interaction mechanism. The trimer complex TaPYR/ABA/TaPP 2C30 formed abrogated the inhibition of TaPP C30 on the common wheat SnRK2 family member TaSnRK2.10. TaSnRK2.10 then interacts with the transcription factor member TaNF-YC1 of the NF-Y family of common wheat, and TaNF-YC1 participates in regulating osmoregulation substance biosynthesis, stomatal movement and cellular reactive oxygen species homeostasis by transcriptional activation TaSLAC-3, taP5CR1, taSOD4, taCAT2 and TaPIN6 expression. The altered physiological processes described above help plants adapt to drought stress. Further identification and functional analysis TaPYR of the molecular processes of the signal pathway involved in TaPYR can provide new insight into drought tolerance mechanisms of plants.

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

In the foregoing embodiments, the descriptions of the embodiments are emphasized, and in part, not described or illustrated in any particular embodiment, reference is made to the related descriptions of other embodiments.

The foregoing embodiments are merely illustrative of the technical solutions of the present invention, and not restrictive, and although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that modifications may still be made to the technical solutions described in the foregoing embodiments or equivalent substitutions of some technical features thereof, and that such modifications or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (1)

1. A primer combination is characterized by being used for detecting single nucleotide polymorphism of SNP loci with physical distance of Ch1A_ 346681497 in a wheat genome version number of Triticum aestivum iwgsc _38310.0, 4 SNP loci of ORF in TaPYR10 correspond to 211, 224, 374 and 410 bases of a sequence shown in SEQ ID NO.1 respectively, when the loci are G/G, T/T, G/G, A/A pure in sequence, the corresponding genotype is Hap1, and when the loci are T/T, A/A, C/C, G/G pure, the corresponding genotype is Hap2; the 5 SNP loci in the promoter region correspond to the 1680 th base, the 1684 th base, the 1685 th base, the 1737 th base and the 1902 th base of the sequence shown in SEQ ID NO. 9 from the last, when the loci are in T/T, A/A, C/C, A/A, G/G homozygosity in sequence, the corresponding genotypes are Hap1, when the loci are in C/C, G/G, T/T, G/G, T/T homozygosity, the corresponding genotypes are Hap2, the proline content, the biomass and the yield are such that the wheat homozygous for the genotype Hap1 is larger than or is candidate larger than the wheat homozygous for the genotype Hap2, and the primer combination is a primer pair consisting of SEQ ID NO. 244-SEQ ID NO.279 in a sequence table.