Wheat ABA Receptor TaPYL5 Constitutes a Signaling Module with Its Downstream Partners TaPP2C53/TaSnRK2.1/TaABI1 to Modulate Plant Drought Response

Abscisic acid receptors (ABR) play crucial roles in transducing the ABA signaling initiated by osmotic stresses, which has a significant impact on plant acclimation to drought by modulating stress-related defensive physiological processes. We characterized TaPYL5, a member of the ABR family in wheat (Triticum aestivum), as a mediator of drought stress adaptation in plants. The signals derived from the fusion of TaPYL5-GFP suggest that the TaPYL5 protein was directed to various subcellular locations, namely stomata, plasma membrane, and nucleus. Drought stress significantly upregulated the TaPYL5 transcripts in roots and leaves. The biological roles of ABA and drought responsive cis-elements, specifically ABRE and recognition sites MYB, in mediating gene transcription under drought conditions were confirmed by histochemical GUS staining analysis for plants harbouring a truncated TaPYL5 promoter. Yeast two-hybrid and BiFC assays indicated that TaPYL5 interacted with TaPP2C53, a clade A member of phosphatase (PP2C), and the latter with TaSnRK2.1, a kinase member of the SnRK2 family, implying the formation of an ABA core signaling module TaPYL5/TaPP2C53/TaSnRK2.1. TaABI1, an ABA responsive transcription factor, proved to be a component of the ABA signaling pathway, as evidenced by its interaction with TaSnRK2.1. Transgene analysis of TaPYL5 and its module partners, as well as TaABI1, revealed that they have an effect on plant drought responses. TaPYL5 and TaSnRK2.1 positively regulated plant drought acclimation, whereas TaPP2C53 and TaABI1 negatively regulated it. This coincided with the osmotic stress-related physiology shown in their transgenic lines, such as stomata movement, osmolytes biosynthesis, and antioxidant enzyme function. TaPYL5 significantly altered the transcription of numerous genes involved in biological processes related to drought defense. Our findings suggest that TaPYL5 is one of the most important regulators in plant drought tolerance and a valuable target for engineering drought-tolerant cultivars in wheat.


Introduction
Drought is one of the most damaging abiotic stresses, having a significant impact on plant growth and crop productivity [1,2]. Crop production has become water-limited in recent years and will be further worsened in the future as a result of global climate change [3,4]. In such cases, the ability of plants to survive in drought conditions is critical for crop growth and development [5,6]. As a result, extensive research on responses of crops to water deficit has become a hotspot for engineering crop cultivars to be high-yielding and drought-tolerant.
Plants have evolved a number of cellular and molecular signaling pathways to activate and regulate plant resistance to osmotic stresses. Numerous studies have been conducted to

TaPYL5 Expression Is Sensitive in Response to Drought Stress
TaPYL5 transcripts were examined during drought treatments to characterize its response to this type of stressor. The TaPYL5 expression levels were significantly upregulated in both roots and aerial tissues in response to drought signaling, displaying a pattern of gradually increasing transcript abundance along with the intensification of drought extent initiated by polyethylene glycol (PEG-6000) with concentrations ranging from 0 to 15% (w/v)) ( Figure 2A). Furthermore, analysis of the TaPYL5 temporal expression pattern

TaPYL5 Expression Is Sensitive in Response to Drought Stress
TaPYL5 transcripts were examined during drought treatments to characterize its response to this type of stressor. The TaPYL5 expression levels were significantly upregulated in both roots and aerial tissues in response to drought signaling, displaying a pattern of gradually increasing transcript abundance along with the intensification of drought extent initiated by polyethylene glycol (PEG-6000) with concentrations ranging from 0 to 15% (w/v)) ( Figure 2A). Furthermore, analysis of the TaPYL5 temporal expression pattern revealed that its transcripts in both tissues mentioned were elevated within a 27-h drought treatment regime, and the drought-induced transcripts were restored following progression of a 27-h normal recovery condition ( Figure 2B). These findings suggest that the TaPYL5 transcription is sensitive to drought stress.

A Suite of Cis-Regulatory Elements in TaPYL5 Promoter Impacts Gene Responses to Drought Stress
A prediction online tool (PLACE) was used to identify the cis-acting regulatory elements located in the TaPYL5 promoter, 2-kb in length upstream of the translation start codon (ATG). The TaPYL5 promoter was found to contain a subset of critical elements, including those involved in RNA polymerase interaction (TATA box), gene expression

A Suite of Cis-Regulatory Elements in TaPYL5 Promoter Impacts Gene Responses to Drought Stress
A prediction online tool (PLACE) was used to identify the cis-acting regulatory elements located in the TaPYL5 promoter, 2-kb in length upstream of the translation start codon (ATG). The TaPYL5 promoter was found to contain a subset of critical elements, including those involved in RNA polymerase interaction (TATA box), gene expression efficiency regulation (CAAT box), and osmotic stress signaling response (i.e., ABRE and DRE elements) ( Figure 2C). Furthermore, the TaPYL5 promoter contains a subset of binding sites for transcription factor MYB proteins, such as MYB88, MYB46, and MYB14, which are frequently involved in gene transcription in response to osmotic stress ( Figure 2D). As a result, these cis-acting regulatory elements may influence the TaPYL5 response to drought at the transcriptional level.
To determine the roles of critical cis-elements in mediating gene transcription efficiency under drought conditions, histochemical GUS staining assays were performed. The results revealed that once driven by truncated promoter fragments lacking distinct elements/element amounts, the expression levels of the GUS reporter were altered. When compared to the staining extent in the control, which was integrated by a full-length (1938 bp) promoter, the GUS staining extent in samples integrated by a set of truncated promoter regions was drastically reduced. This indicates that the GUS staining extent is closely correlated with the promoter length integrated under drought conditions. Although reduced GUS staining extents were detected in samples driven by truncated promoter regions, all of the staining extents in leaf samples harboring TaPYL5pro378-GUS (D1, −378), TaPYL5pro640-GUS (D2, −640), TaPYL5pro1055-GUS (D3, −1055), TaPYL5pro 1635-GUS (D4, −1635), and TaPYL5pro1938-GUS (D5, −1938) treated by drought displayed stronger GUS staining and activities than the control (D5) cultured under normal growth conditions ( Figure 2E). These results suggest that the cis-acting regulatory elements located at various positions in the promoter, such as elements ABRE and DRE and recognition sites of MYB, exert positive functions in mediating the transcriptional response of TaPYL5 to drought stress.

TaPYL5 Involves the Constitution of an ABA Core Signaling Module with PP2C and SnRK2 Members
The TaPYL5-involved establishment of an ABA core organelle module was determined by yeast two-hybrid and BiFC assays. The yeast two-hybrid assay revealed that the TaPYL5 protein interacted specifically with a PP2C member known as TaPP2C53 ( Figure 3A). The TaPP2C53 protein specifically interacted with TaSnRK2.1, a member of the SnRK2 family in wheat, according to further analysis using a similar strategy for identifying TaPYL5-TaPP2C53 protein interaction ( Figure 3B). Therefore, this suggests that TaPYL5 establishes an ABA core signaling module with its downstream partners, namely TaPYL5-TaPP2C53-TaSnRK2.1 that is thought to transduce drought-induced ABA signaling and to mediate plant drought response.
The BiFC analysis confirmed the protein-protein interactions observed in the yeast twohybrid assay. As expected, strong YFP signals derived from TaPYL5-nYFP, TaPP2C53-cYFP, TaPP2C53-cYFP and TaSnRK2.1-nYFP were detected in leaves of the Nicotiana benthamiana model plant system co-transformed with each of the above cassette combinations ( Figure 3C). In summary, TaPYL5 participates in the establishment of an ABA core signaling module with its downstream partners, which transduces the ABA signaling and mediates the plant response to drought stress by regulating the related physiological processes. The BiFC analysis confirmed the protein-protein interactions observed in the yeast two-hybrid assay. As expected, strong YFP signals derived from TaPYL5-nYFP, TaPP2C53-cYFP, TaPP2C53-cYFP and TaSnRK2.1-nYFP were detected in leaves of the Nicotiana benthamiana model plant system co-transformed with each of the above cassette combinations ( Figure 3C). In summary, TaPYL5 participates in the establishment of an ABA core signaling module with its downstream partners, which transduces the ABA signaling and mediates the plant response to drought stress by regulating the related physiological processes.

Transgenic Lines of Genes Encoding TaPYL5 and Its Downstream Partners Modify Plant Drought Response
TaPYL5 and its downstream partner genes were subjected to transgene analyses in wheat in order to characterize their functions in response to drought stress. The transgenic lines of TaPYL5 (Sen 2, Sen 3, Anti 1, and Anti 2), TaPP2C53 (Sen 1, Sen 2, Anti 1, and Anti 2), and TaSnRK2.1 (Sen 3, Sen 4, Anti 2, and Anti 3) with typical overexpression or knockdown of the target (Supplementary Figure S2), were subjected to normal growth and drought treatments. Under normal growth conditions, all of these transgenic lines exhibited similar phenotypic behaviors, tissue biomass both aboveground and root, and root volume with the wild-type (WT) (Figures 4 and 5A-D). Under drought treatments, the transgenic lines were modified for the above growth traits, with improved growth and increased biomass of aboveground and root tissues, as well as elevated root volumes, observed in lines overexpressing TaPYL5 (Sen 2 and Sen 3), and inhibited growth, decreased biomass, and lowered root volumes observed in lines with TaPYL5 knockdown expression (Anti 1 and Anti 2) compared to the WT (Figures 4 and 5A

TaPYL5-Mediated Drought Response Is Associated with the Role in Modulating Stomata Movement, Osmolyte Accumulation, and ROS Homeostasis
The osmotic stress-related physiological indices, such as stomata closing rate (SCR), leaf water losing rate (WSR), proline and soluble sugar contents, and ROS homeostasis parameters, were analyzed for the TaPYL5, TaPP2C53, and TaSnRK2.1 lines. In accordance with the plant growth traits, the TaPYL5 lines (Sen 2, Sen 3, Anti 1, and Anti 2) were found to be similar on the stress-related indices tested, demonstrating comparable SCR in a 2 h drought treatment regime, WSR in a 3 h water deficit condition. Similarly, under normal conditions, the contents of proline and soluble sugar, as well as the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA), were compared to the WT. Under drought conditions, the TaPYL5-overexpressing lines (Sen 2 and Sen 3) exhibited improved SCR ( Figure 6A,B), decreased WLR ( Figure 6C), increased osmolytes (proline and soluble sugar) ( Figure 6D,E), enhanced SOD, POD, and CAT activities ( Figure 6F-H), and decreased MDA accumulation ( Figure 6I). In contrast to the preceding lines, those with TaPYL5 knockdown expression (Anti 1 and Anti 2) demonstrated the opposite behavior on those indices ( Figure 6A-I). The results of the above osmotic stress-related physiological indices of TaPP2C53 (Sen 1, Sen 2, Anti 1, and Anti 2) and TaSnRK2.1 (Sen 3, Sen 4, Anti 2 and Anti 3) lines were also consistent with their growth characteristics. According to the physiological indices obtained from transgene analyses, the TaPYL5-involved establishment of an ABA core organelle module mediates plant drought tolerance, which is closely associated with the modulated stress responsive-associated physiological processes.

TaSnRK2.1 Protein Interacts with ABA-Responsive Transcription Factor TaABI1 to Negatively Impact Plant Drought Resistance
To better understand the molecular process underlying the ABA signaling modulemediated plant drought response, the putative downstream partner(s) of TaSnRK2.1 were identified using a yeast two-hybrid assay. TaABI1, an ABA responsive-associated transcription factor, was found to interact with TaSnRK2.1 specifically ( Figure 7A). Further BiFC assays confirmed the interaction between TaSnRK2.1 and TaABI1; strong YFP signals were detected in Nicotiana benthamiana explants co-transformed by TaSnRK2.1-nYFP and TaABI1-cYFP ( Figure 7B). These findings suggested that TaABI1 functions as a downstream partner in the TaPYL5-involved module regulation, potentially contributing to plant drought response.
Transgenic lines with modified TaABI1 expression were created in order to better understand its role in mediating plant drought response. Lines OE 1 and OE 2, two wheat TaABI1overexpressing lines, and KE 1 and KE 2, two wheat TaABI1 knockdown lines that were specified by target overexpression or knockdown expression (Supplementary Figure S3), were subjected to drought treatment. Results showed that they were heavily influenced by growth traits and osmotic stress-related physiological indices, such as SCR, WSR, and contents of oymolytes. The plant growth was inhibited and the related physiological indices (i.e., decreased SCR, increased WLR, and proline and soluble sugar contents) were decreased in lines OE 1 and OE 2, whereas KE 1 and KE 2 had the opposite effect on the traits shown in the above lines with TaABI1 overexpression ( Figure 7C-H). These findings suggest that TaABI1 acts as a regulator downstream of TaSnRK2.1 to negatively regulate plant drought adaptation.

Transcriptome Profile Mediated by TaPYL5 upon Drought Signaling
The effects of TaPYL5 on the transcriptome were investigated using an RNA-seq analysis on a drought-stressed TaPYL5-overexpressing line (Sen 1) and the WT plants as samples. The results revealed that 1669 genes were differentially expressed in Sen 1 compared to the WT, with 610 upregulated and 1059 downregulated (Supplementary datasets S1 and S2). To verify the reproducibility of transcriptome results, qRT-PCR was performed on ten differentially expressed genes (DEGs), five of each of which were upregulated or downregulated in expression. The results on transcripts detected from all of them were consistent with those shown in the RNA-seq analysis, namely, the expression levels of the five upregulated genes (TaPT5, TaLD2, TaWRKY9, TaNPP, and TaF3H) were increased, whereas those of the other five DEGs with downregulated expression (TaWAT1, TaADH2D, TaSADP, TaCBF, and TaH2B) were decreased in Sen 1 with respect to the WT (Supplementary Figures S4 and S5). The qRT-PCR results for these DEGs validated the transcriptome pattern underlying TaPYL5 modulation in response to drought stress.
Gene ontology (GO) analysis on the DEGs classified them into the GO terms associated with 'biological process', 'molecular functions', and 'cellular component'. Thirteen subgroups of DEGs are overrepresented by functions associated with 'biological process', including cellular processes and metabolic processes, among others. The DEGs in the term 'molecular function' include seven subgroups that are functional in molecule binding, modulating catalytic activity of enzymes, and regulating transcription regulator activity, etc. The DEGs in the term 'cellular component' include 12 subgroups that are effective in affecting cell part, organelle, and membrane part, among other things ( Figure 8A). The DEG-modulated biochemical pathways were further defined by the KEGG analysis. Over 50 pathways underlying TaPYL5 modulation are proposed to have been altered, particularly those associated with ion trafficking and homeostasis, trivalent inorganic anion homeostasis regulation, and glucosamine-containing compound metabolism, among others ( Figure 8B). These findings suggest that TaPYL5 has a broad impact on regulating plant drought response by modifying gene transcription at the global level.

TaPYL5 Response to Drought Signaling Associates with a Set of Cis-Elements Situated in Promoter
While exposed to stressors, the transcription efficiencies of genes in the ABA signaling pathway are altered [30][31][32]. Investigations into members of the ABA receptor family have documented their responses to osmotic stress at the transcriptional level. For example, expression analysis of AtPYL, a PYR member of A. thaliana, revealed that it was upregulated on the transcript abundance under drought and high salinity conditions, which led to altered transcription of a subset of stress defensive-associated genes and improved plant acclimation to the above stressors [33]. Our analysis of TaPYL5 expression patterns during drought signaling revealed that it is sensitive to drought stress; more TaPYL5 transcripts were detected in roots and aerial tissues under intensified stress extent and the progress of stress duration. This finding suggests that it plays a role in transducing drought signaling, impacting on plant stress response. So far, a set of cis-acting regulatory elements in promoters that modulate plant response to ABA and drought signaling has been identified. Of these, the elements ABRE (specified by motifs ACGTG, AACCCGG, and CGTACGT-GCA) [34] and the transcription factor recognition and binding sites, such as MYB, bZIP, and MYC members, all contribute to gene response to drought stress at the transcriptional level [35]. We identified the cis-acting elements responsive to ABA and drought through an online search of the TaPYL5 promoter. Further GUS histochemical staining assays to define reporter expression under control of the TaPYL5 promoter fragments, which included or lacked the aforementioned distinct cis-elements, confirmed their positive roles in regulating gene transcription efficiency. Further characterization of these cis-acting elements using base mutation technology can help better understand the mechanism of TaPYL5's drought response.

TaPYL5 Is Involved in Establishing an ABA Signaling Module with Distinct PP2C and SnRK2 Members to Contribute to Plant Drought Response
The ABA core signaling module, which is made up of different ABR members and its downstream partners, plays critical roles in signaling transduction, and its dissection aids greatly in understanding the mechanism of module-mediated plant acclimation to drought stress [36]. Our previous investigation into TaPYL4, a member of the ABA receptor family, indicated that it is involved in the constitution of an ABA core signaling module with PP2C member TaPP2C2 and SnRK2 member TaSnRK2.1 [37]. This study was initiated to better understand the ABA core signaling module composed of a TaPYL5 protein and PP2C member, as well as the PP2C protein interacting protein(s) in the SnRK2 family. The yeast two-hybrid and BiFC assays were used to identify a PP2C member, TaPP2C53, and a SnRK2 protein, TaPP2C.1, both of which are involved in the establishment of an ABA core signaling module. As a result, this module TaPYL5/TaPP2C53/TaSnRK2.1 is proposed to be functional in transducing drought-induced signaling in wheat. ABR and its downstream partners' interaction and action mechanisms were previously defined [38]. The ABR protein, for example, changes its conformation and then binds ABA induced by osmotic stresses, and the mobile gating loop established after ABA binding allows it to interact with a distinct clade A PP2C protein by interacting with the conserved active site [38,39]. Our protein analysis on the three-dimensional structure revealed conserved motifs in the TaPYL5 protein, implying that they are potentially involved in the proteinprotein interaction process. However, detailed molecular mechanisms of how the TaPYL5 protein interacts with its partner, TaPP2C53, need to be elucidated. Further research on the dynamic molecule coupling between TaPYL5 and TaPP2C53, as well as the latter and its downstream partner, TaSnRK2.1, can help us understand the action mechanism of the ABA core signaling component in wheat. However, the detailed molecular mechanisms by which the TaPYL5 protein interacts with its partner, TaPP2C53, need to be determined.

TaPYL5 and Genes Encoding Its Partners Contribute Largely to Plant Drought Response
Functional characterization of ABR family members has revealed that they are extensively involved in mediating diverse physiological processes in plants, such as seed dormancy, germination, lateral root formation, light signaling convergence, and flowering time control [40][41][42]. Further investigations have also confirmed their roles in promoting plant acclimation to osmotic stresses. For example, studies into AtPYL5, AtPYL8, and AtPYL9, three members of the PYL family in A. thaliana, revealed that they act as crucial regulators in mediating plant response to exogenous ABA during seed germination and seedling growth, promoting stomatal movement and improving plant stress resistance [43][44][45]. A number of the genes of the PYL family including OsPYL3, OsPYL5, and OsPYL9 in rice (Oryza sativa), GhPYL10, GhPYL12 and GhPYL26 in cotton (Gossypium herbaceum), and ZmPYL3, ZmPYL9, ZmPYL10 and ZPYL13 in maize (Zea mays), enhance the plant response to ABA under osmotic stress conditions, positively modulating osmolyte accumulation, and improving plant biomass production ability [46][47][48]. In wheat, previous investigations into TaPYL4 have indicated that it acts as one of the critical regulators in positively regulating plant drought tolerance [37,49]. The transgenic wheat lines with TaPYL4 overexpression displayed increased plant ABA sensitivity, lowered water consumption during the plant's lifetime by reducing transpiration, increased photosynthetic activity, and improved wateruse efficiency under water deficit conditions [49]. To characterize the functions of TaPYL5, TaPP2C53, and TaSnRK2.1 in mediating plant drought response, we generated transgenic wheat lines for TaPYL5, TaPP2C53, and TaSnRK2.1. The lines for target genes with overexpression or knockdown expression showed significant changes in growth behaviors, such as phenotypes and plant biomass. TaPYL5 and TaSnRK2.1 positively and TaPP2C53 negatively regulated plant adaptation to drought stress. These results from TaPYL5 and its partners confirmed the importance of the TaPYL5/TaPP2C53/TaSnRK2.1 action module in transducing drought signaling via an ABA-dependent pathway and regulating plant drought response.
Drought adaptation of plants is closely related to stress-related physiological processes, such as stomata movement that is adapted to closing to avoid water loss by transpiration [50,51], induction of osmotic substance accumulation [52], and enhancement of ROS homeostasis by scavenging ROS via antioxidant enzyme capacity enhancement [53,54]. Our investigation into TaPYL4 revealed that this wheat-ABA-receptor gene-mediated plant drought response is associated with its role in transcriptionally regulating TaPIN9, a PIN-FORMED gene modulating cellular auxin translocation. The modified expression of this PIN-FORMED member improves the establishment of root system architecture (RSA) underlying TaPYL4 regulation to contribute to plant drought adaptation [37]. In this study, we investigated the physiological parameters related to plant osmotic stress response to better understand plant drought responses mediated by the TaPYL5-involved ABA signaling pathway. Lines overexpressing TaPYL5 and TaSnRK2.1 showed increased SCR when challenged by drought signaling, increased water retention capacity in droughttreated detached leaves, elevated osmolytes (i.e., proline and soluble sugar contents), and increased AE activities (i.e., SOD, CAT, and POD). Lines with TaPP2C53 overexpression, on the other hand, displayed opposite patterns on the above indices to those seen in the TaPYL5 and TaSnRK2.1 lines. The plant growth behaviors underlying TaPYL5 modulation and its partners constituting an ABA core signaling module are consistent with the protein-protein interaction mechanism involved in drought-initiated signaling. As a result, the signaling module composed of PYL, PP2C, and SnRK2 members in wheat, namely TaPYL5/TaPP2C53/TaSnRK2.1, is functional in plant drought acclimation by its large impact on stress-related physiological processes.

TaABI1 Is a Downstream Component of TaPYL5 Signaling Module and Negatively Regulates Drought Adaptation
Members of the ABA responsive transcription factor family (ABI) modulate gene transcription in response to exogenous ABA and act as a critical regulator in plant osmotic stress response, affecting a subset of stress-responsive-related physiological processes [55]. We investigated whether any member of the TaABI family connects the TaPYL5-involved module in plant drought response by analyzing putative protein-protein interactions between TaSnRK2.1 and a member(s) of the TaABI family. The yeast two-hybrid and BiFC protein-protein interaction assays revealed that TaABI1 specifically interacted with TaSnRK2.1. This finding suggests that TaABI1 may be involved in drought signaling transduction via the ABA core signaling pathway established by TaPYL5 and its partners. The transgenic wheat lines of TaABI1 showed modified growth behaviors (phenotypes and plant biomass), altered physiological indices such as stomata movement and osmolytes (proline and soluble sugar contents) with respect to the WT under drought treatment; plant drought adaptation was reduced in lines with TaABI1 overexpression, whereas plant drought tolerance was improved in lines with gene knockdown. These findings establish a link between the TaPYL5-involved ABA signaling module and its downstream partner TaABI members, which build the TaPYL5/TaPP2C53/TaSnRK2.1-TaABI1 signaling pathway to act cooperatively in regulating plant drought response.

TaPYL5 Modifies Gene Transcription Globally to Impact Plant Drought Response-Associated Physiological Processes
It has been shown that ABA signaling induced by osmotic stressors causes a significant change in the expression of a number of genes involved in mediating plant osmotic stress response, thereby altering stress-related physiological parameters and biochemical pathways [56,57]. To gain a better understanding of the molecular processes underlying the TaPYL5-mediated plant drought response, we used RNA-seq to identify the genes with altered transcription efficiency under drought conditions. A large number of genes on transcripts in the transgenic line with TaPYL5 overexpression (Sen 1) were significantly altered when compared to the drought stressed WT plants. According to KEGG, they regulate over fifty biochemical pathways, with a focus on ion trafficking and homeostasis, trivalent inorganic anion homeostasis regulation, and glucosamine-containing compound metabolism. The transcriptome profile analysis suggests that TaPYL5 has a broad impact on modulating gene transcription that regulates plant acclimation/adaptation to drought stress. Based on our findings, we proposed a model for TaPYL5, an ABR member in wheat, to mediate plant drought response (Figure 9). TaPYL5 transcripts are induced in plant roots and aerial tissues in response to drought signaling. After binding ABA molecules, the induced TaPYL5 proteins acclimatize to interact with TaPP2C, a member of the clade A phosphatase (PP2C), via a protein-protein interaction mechanism. The formation of this trimer complex molecule (TaPYL5/ABA/TaPP2C53) eliminates TaPP2C53's inhibition of TaSnRK2.1, a member of the SnRK2 family in wheat. Then, TaSnRK2.1 interacts with TaABI1, an ABA-responsive TF in wheat that extensively modulates transcription of genes involving regulation of osmotic stress-associated physiology, such as stomata movement, osmolytes biosynthesis, and cellular ROS homeostasis, to contribute to plant adaptation to drought stress. Further identification and functional dissection of unknown components acting as members of the TaPYL5-involved signaling pathway may provide insights into the mechanism of plant osmotic stress resistance.

Characterization Analysis on TaPYL5
TaPYL5 (GenBank accession No. TraesCS1A02G126800), a gene in wheat with a high nucleic acid sequence similarity to ABA receptor (PYL) members in our previous RNAseq analysis (unpublished data), was found to be significantly upregulated in expression during drought stress. The open read frame (ORF) and a 2 kb promoter region of TaPYL5 situated on chromosome 1D (TaPYL5-1D, hereafter named TaPYL5 for simplicity) and

Characterization Analysis on TaPYL5
TaPYL5 (GenBank accession No. TraesCS1A02G126800), a gene in wheat with a high nucleic acid sequence similarity to ABA receptor (PYL) members in our previous RNA-seq analysis (unpublished data), was found to be significantly upregulated in expression during drought stress. The open read frame (ORF) and a 2 kb promoter region of TaPYL5 situated on chromosome 1D (TaPYL5-1D, hereafter named TaPYL5 for simplicity) and those of its homeologs located on chromosomes 1A (TaPYL5-1A, TraesCS1B02G145800) and 1B (TaPYL5-1B, TraesCS1D02G126900) were obtained from Ensembl Plants (http://plants.ensembl.org/ index.html accessed on 22 April 2023), which have a high degree of similarity at the nucleic acid and amino acid levels (98.18-98.51%, Supplementary Figure S6A,B). TaPYL5, in comparison to other homeologs, showed significantly induced transcripts in response to drought stress (Supplementary Figure S6C). Conserved domains of TaPYL5 protein were defined by plant PYL counterparts as previously described [58]. Phylogenetic tree covering TaPYL5 and its homologous genes across various plant species were established using the MegAlign algorithm supplemented in DNAStar software (www.dnastar.org accessed on 22 April 2023). The sequences used in phylogenetic relation analysis were obtained with a BLASTn search against the National Center for Biotechnology Information's (NCBI) GenBank database (www.https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 12 January 2022).

Analysis of TaPYL5 Protein Localization at the Subcellular Level
An experiment was carried out in order to determine the subcellular localization of the TaPYL5 protein after endoplasmic reticulum (ER) assortment. The ORF of TaPYL5 was amplified using gene specific primers (Supplementary Table S1) using a reverse transcriptionpolymerase chain reaction (RT-PCR) and then integrated into the binary vector pCAM-BIA3300 in a frame with the reporter gene, a green-fluorescent-protein encoding gene (GFP), downstream of the CaMV35S promoter. The expression cassette TaPYL5-GFP was genetically transformed onto the Agrobacterium tumefaciens strain EHA105 using the standard heat-shock method. Following the A. tumefaciens-mediated transformation method, the transformants containing the cassette were used to genetically transform epidermal cells of N. benthamiana. After 48 h of infiltration, the GFP signals initiated by the fusion of TaPYL5-GFP, as well as those initiated by the control CaMV35S-GFP integrated by an empty vector, were detected under a fluorescent microscope, as previously described [59]. Furthermore, a protoplast expression system of N. benthamiana was used to verify the location of TaPYL5 at the cellular level, as previously described [60,61]. Briefly, protoplasts were firstly obtained from young leaves of N. benthamiana [60]. They were then transformed using the above expression cassettes containing TaPYL5-GFP and a modified PEG-mediated transformation protocol [61]. After 24 h of infiltration between A. tumefaciens transformants and explant cells, GFP signals initiated in protoplasts transformed by the fusion were detected under a fluorescent microscope.

Expression Analysis of TaPYL5 upon Drought and Characterization of Cis-Acting Regulatory Elements in the TaPYL5 Promoter
The expression patterns of TaPYL5 in response to drought stress were studied using seedlings of Shimai 26, a drought-tolerant cultivar cultured in a modified MS solution supplemented with an osmotic-regulatory chemical (i.e., PEG-6000). Seeds were germinated on a regular basis, and seedlings were cultured in a standard MS solution, as previously described [62]. They were subjected to a simulated drought treatment at the second-leaf stage by growing in a modified MS solution supplemented with various concentrations of PEG-6000, including 0, 1, 5, 10, and 15% (w/v), corresponding to osmotic potentials of 0, −0.35, −0.81, −1.08, and −1.27 mPa, respectively. Root and leaf samples were collected over a 27-h period of the treatments, with time points of 1, 3, 9, and 27 h during drought conditions. A normal recovery treatment was established by re-growing the droughtchallenged seedlings in a standard MS solution after 27 h to define the gene response to recovered normal conditions. Root and leaf tissues were collected at 1, 3, 9, and 27 h after the recovery treatment. TaPYL5 transcripts in samples collected during drought and recovery treatments were assessed using qRT-PCR using gene specific primers (Supplementary Table  S1), as previously described [63]. Tatubulin, a constitutive gene of wheat, was used as an internal reference to normalize the target transcripts during this time.
An online search tool referred to as PLACE (http://bioinformatics.psb.ugent.be/ webtools/plantcare/html/ accessed on 12 January 2022) was adopted to identify the putatively critical cis-acting regulatory elements situated in the TaPYL5 promoter. A suite of promoter regions with truncated sizes across 2 kb upstream ATG was used to functionally characterize a set of the elements in modulating transcription efficiency on the gene under drought conditions (translation start codon). PCR amplified sequences with lengths of 378, 640, 1055, 1635, and 1938 bp that contained various elements/amounts associated with gene transcription regulation and osmotic stress response (i.e., ABRE, and recognition sites MYB) (Supplementary Table S2) were separately inserted in front of reporter GFP in binary vector pCAMBIA3301 [64]. Transformants of A. tumefaciens strain EHA105 containing different cassettes were used to transform embryos of Shimai 22, as previously described [65]. Independent transgenic lines containing each construct were germinated on a regular basis, and seedlings were grown normally in an MS nutrient solution to the third leaf stage. They were then subjected to a 6 h simulated drought treatment regime by culturing in a modified MS solution supplemented with 10% (w/v) of PEG-6000, an osmotic-regulatory chemical for drought induction. GUS activities in transgenic lines and controls were assessed using histochemical staining for 24 h at 37 • C using GUS staining solution, which contained the following constituents: 0.1% Triton X-Gluc, pH 7.2, and 10 mM EDTA. After several wash processes with graded ethanol series, GUS staining results in samples were documented using a digital camera.

Yeast Two-Hybrid and BiFC Assays to Identify Protein-Protein Interactions
Yeast two-hybrid assays were carried out to identify the partner(s) of PP2C interacted with by the TaPYL5 protein as well as any SnRK2 family member(s) acting as the downstream partner of the PP2C member. Six PP2C family genes and seven SnRK2 family genes in wheat were identified using the NCBI GenBank database. The PP2C family members subjected to assay included TaPP2C6-3B, TaPP2C8-1A, TaPP2C9-3B, TaPP2C50-1D, and TaPP2C53-1A (simplified to TaPP2C6, TaPP2C8, TaPP2C9, TaPP2C50, and TaPP2C53, respectively); the SnRK2 family genes subjected to assay included TaSnRK2 Table S1 contains information on the genes mentioned in the PP2C and SnRK2 families. During this assay, the TaPYL5 protein expressed in yeast strain was used as bait, while the proteins in the PP2C family were expressed separately as preys. To elicit the putative protein interaction process, yeast transformants expressing TaPYL5 protein and each TaPP2C member in the host strain (AH109) were co-cultured in a selecting solid medium supplemented with exogenous ABA (1 µM). Similarly, the TaPP2C member that interacted with the TaPYL5 protein identified above was used as prey, while the proteins in the SnRK2 family were used as bait separately. The above yeast two-hybrid assays were carried out in accordance with the manufacturer's instructions [63]. Supplementary Table S1 shows the gene specific primers used for amplification of TaPYL5 and the genes mentioned in the PP2C and SnRK2 families.
BiFC assays were used to validate protein interactions between TaPYL5 and PP2C members, as well as the PP2C protein and SnRK2 family members. The ORFs of target genes, including TaPYL5 and members of the PP2C and SnRK2 families, were amplified by RT-PCR using gene specific primers (Supplementary Table S1) and integrated in frame with fragments of the reporter gene YFP (yellow fluorescent protein encoding gene) at the C-or N-terminus, as previously described [66]. TaPYL5 and TaPP2C2 expression cassettes, as well as TaPP2C and TaSnRK2 expression cassettes, were transiently co-transformed onto N. benthamiana leaves using the Agrobacterium (stain EHA105)-mediated infiltration method [65]. Following the manufacturer's recommendation, YFP signals in transgenic leaves were detected after 2 d of infiltration using a fluorescent microscope (Olympus FV10-ASW, Tokyo, Japan). Parallel to the detection of YFP signals, DAPI (Solarbio, Beijing, China) staining was used as a positive control to demonstrate nucleus targeting, as previously described [67].

Assessment for Growth Traits of Transgenic Lines
Transgene analysis on TaPYL5 and genes encoding its partners (TaPP2C53, TaSnRK2.1, and TaABI1) was conducted to evaluate their roles in mediating plant drought response. TaPYL5, TaPP2C53, TaSnRK2.1, and TaABI1 ORFs were amplified in both sense and antisense orientations using RT-PCR and gene specific primers (Supplementary Table S1). They were then separately inserted into the NcoI/BastEII sites of the binary vector pCAMBIA3301, which was controlled by the CaMV35S promoter. Transformants of A. tumefaciens strain EHA105 harboring the above expression cassettes were subjected to transformation onto Shimai 22 wheat as described previously [66]. For each gene, a set of five to six transgenic lines in either sense or antisense orientation was created. qRT-PCR with gene specific primers (Supplementary Table S1) was used to assess the expression levels of the target gene in transgenic lines. To address gene functions in modulating plant drought response, two lines were selected at the T3 generation for each gene with more target transcripts and another two with lower expression levels on target genes, with one copy insertion each at the genome level. Briefly, seeds from transgenic lines and the WT were germinated on a regular basis, and seedlings were cultured in plastic pots filled with a mix of vermiculate and fertile soil (1:1, w/w) under normal growth conditions supplied by tap water daily to maintain 70-75% relative soil moisture. They were then subjected to drought treatment at the second-leaf stage by controlling the water supply to maintain 55-60% of relative soil moisture detected by a soil water potential metre (TRS-IIN, Shanghai, China). Growth traits in transgenic lines, including phenotypes, biomass in aerial tissues and roots, and root volumes, were evaluated three weeks after treatments. The phenotypes were documented using digital camera images, while the biomass of plant tissues and the volumes of root tissues were obtained using conventional assay methods.

Determination of Physiological Parameters Associated with Osmotic Stress Responses in Transgenic Lines
After normal growth and drought treatment, a subset of the physiological indices associated with plant osmotic stress responses was measured in upper expanded leaves of the transgenic wheat lines. The SCR, WLR, osmolytes contents, and ROS homeostasisrelated parameters were all evaluated. The SCR values were calculated by measuring stomata aperture on the epidermis at the indicated time points (0, 0.5, 1, and 2 h) during a 2 h regime of simulated drought treatment for the third leaf stage seedlings, as previously described [67]. The WLR values were calculated based on decreased fresh weights in upper-expanded leaves at the indicated time points (0.25, 0.5, 1, 2, and 3 h) during a water deficit stressor initiated by putting on a clean bench in comparison to that assessed at time point 0 h (prior to treatment), as previously described [68]. The contents of two types of osmolytes, including proline and soluble sugar, were determined using methods described previously [67]. Among the ROS-associated parameters, antioxidant enzyme activities of SOD, catalase CAT, and POD), and MDA in transgenic and the WT leaves were determined using methods described previously [69].

Transcriptome Analysis
RNA-seq analysis was carried out using the 24 h drought (10% PEG, w/v)-stressed TaPYL5 transgenic line (Sen 2) and the WT roots as samples. Briefly, the total RNA in root tissues of Sen 2 and WT was extracted after drought treatment using the TRIzol reagent (Invitrogen, Waltham, MA, USA). They were then used to construct strand-specific RNAseq libraries in triplicates using the previously described method [63]. The transcripts in the libraries were sequenced on an Illumina HiSeq 2500 sequencing platform (Illumina, San Diego, CA, USA). Raw reads generated from the libraries were further processed using Trimmomatic after adaptors were removed from the reads and the reads were classified as low-quality [70]. The reads generated from the transgenic line (Sen 2) and the WT were then subjected to systematical sequence alignment analysis by searching against the T. aestivum reference transcript database (Novogene Co., Ltd., Beijing, China). Differentially expressed genes were identified in drought-stressed Sen 2 roots using a two-fold modified transcript abundance compared to the WT, calculated by the edger program, which effectively compares read occurrence frequencies between two group samples [71]. During this time, raw P values were calculated with a false discovery rate (FDR) of less than 0.05, as previously suggested [72]. Plant MetGenMap, a web-based tool (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi accessed on 10 May 2022), was adopted to characterize the pathways of KEGG for the DE genes identified in the transgenic line (Sen 2), using the pearl module program CPAN [73].

Statistical Analysis
Gene transcripts, growth traits, plant biomass, and osmotic stress-related indices were all averaged from triplicate data. The Statistical Analysis System software (SAS Corporation, Cory, NC, USA) was used to calculate standard errors for averages and statistical significance analyses for the traits studied. The significance between treatments was compared using Student's t-test with the least significant difference (LSD) assessed at the level of p < 0.05.

Conclusions
TaPYL5 shares conserved domains with plant ABR proteins and is targeted to the stomata, plasma membrane, and nucleus following an ER selection. TaPYL5 expression is drought-sensitive, and its transcription is determined significantly by the droughtresponsive cis-acting regulatory elements located in the gene promoter. TaPYL5 and its two downstream partners, TaPYL5/TaPP2C53/TaSnRK2.1, were constituted to be the ABA core signaling module in yeast two-hybrid and BiFC assays. Wheat transgene analysis revealed that TaPYL5 and TaSnRK2.1 positively regulate plant drought tolerance while TaPP2C53 negatively regulates it by modulating stress-related physiological indices such as stomata movement, osmolytes biosynthesis, and cellular ROS homeostasis. TaABI1 interacts with TaSnRK2.1 specifically and negatively regulates plant adaptation to drought stress, implying that it is a downstream component of the TaPYL5-involved signaling pathway. TaPYL5 plays a significant role in global gene transcription regulation, with a focus on three GO terms that lead to plant physiological adaptation to drought conditions. TaPYL5 and its downstream partners are potential targets for molecular breeding for drought-tolerant cultivars in wheat.