Characterization of TbPYL gene family
Using the above methods, 15 PYL genes were identified in T. boeoticum. The protein lengths range from 176 (TbPYL4) to 232 (TbPYL14) aa, molecular weights from 18.89 (TbPYL5) to 24.97 (TbPYL14) kDa, and isoelectric points from 4.5 (TbPYL4) to 8.99 (TbPYL13). Among them, 66.67% of the proteins are acidic (pI < 7). The instability index ranges from 27.84 (TbPYL7) to 58.76 (TbPYL12), and the aliphatic index ranges from 70.22 (TbPYL7) to 90.24 (TbPYL12). Except for proteins TbPYL2, TbPYL5, and TbPYL8, all others are hydrophilic (GRAVY < 0) (Table.S1, Fig. 1). These genes are unevenly distributed across 5 chromosomes in T. boeoticum, with 6 PYL genes identified on chromosome 3Ab, while chromosomes 5Ab and 6Ab do not harbor any PYL genes (Fig. 2a). Subcellular localization prediction shows that identified PYL proteins localize to both the cytoplasm (53.3%) and chloroplasts (46.6%) (Table.S1).
Analysis of phylogenetic relationships of TbPYLs
To investigate the evolutionary relationships of PYL genes, we constructed a phylogenetic tree using 15 PYL amino acid sequences from T. boeoticum, alongside PYL proteins from A. thaliana and wheat, totaling 72 proteins. Based on sequence similarity, all sequences are divided into 3 subfamilies (Fig. 2b). Specifically, Group Ⅱ has the most members, containing 8 genes; Group Ⅲ has only 2 members, and Group Ⅰ has 5 members.
Gene structure and conserved motifs analysis
Comparative analyses of exon-intron structures can reveal conserved and divergent features, providing clues about the evolutionary history and functional diversification of gene families. The sequence logo indicates that the PYL gene family has high protein sequence similarity, especially within the red box area (Fig. 3a). Our findings indicate that the exon-intron structure of the wheat PYL genes varies across the different subfamilies. Specifically, genes in Group Ⅰ and Ⅱ have a single exon, while those in Group Ⅲ, TbPYL2, and TbPYL14 contain three exons (Fig. 3b). Interestingly, TbPYL11 is an exception, as it contains two exons. To identify conserved motifs within the TbPYL family, we analyzed the full-length protein sequences of 15 TbPYLs using the MEME website. Our analysis revealed a total of 7 distinct motifs. Motif 1 is common to all TbPYL genes, while motifs 2 and 3 are shared by Group Ⅱ and Ⅲ. For Group Ⅰ, motif 4 and 6 is specific. Additionally, motif 5 is present in all members of Group Ⅲ and in Group Ⅱ members except for TbPYL9 and TbPYL15 (Fig. 3b).
Promoters analysis of TbPYL genes
To further understand the transcriptional regulation and potential functions of TbPYL genes, the cis-regulatory elements in the promoters were predicted. This analysis identified a total of 46 distinct cis-elements within the 2000 bp upstream region from the transcription start sites of the TbPYL genes, in addition to the core cis-elements (Table.S2). These cis-elements play important roles in biological processes such as stress responses, hormone responses, metabolic regulation, and growth and development. Among the identified elements, transcription factor binding are the most numerous, followed by light responsive elements and stress responsive elements (Fig. 4a). G-box is the most abundant light-responsive element, present in all TbPYLs. All PYL genes contain at least one hormone responsive element. Among them, abscisic acid responsive element (ABRE) is the most abundant element, with TbPYL8 containing as many as 14 ABRE elements (Fig. 4b), which is consistent with the function of PYL as an abscisic acid receptor. Meanwhile, auxin-responsive elements (AuxRR-core, TGA-element), MeJA-responsive (CGTCA-motif, TGACG-motif), gibberellin-responsive (GARE, P-box), and salicylic acid-responsive elements (TCA-element) were found in TbPYL genes. Additionally, it contains various types of stress response elements, such as anoxic specific inducibility (GC-motif), low-temperature-responsive (LTR) elements and drought-inducibility (MBS), defense, and stress responsiveness (TC-rich repeats). The findings suggest that TbPYL genes may play a role in the transcriptional control of plant growth, hormone regulation, and stress responses.
Prediction of protein 3D structure
In this study, three-dimensional structural homology modeling was performed on the amino acid sequences of 15 TbPYL gene family members from T. boeoticum (Fig. 5a). The online software Phyre2 analysis indicated that all 15 TbPYL proteins exhibited α-helices and β-sheets as their main structural elements. For further verification, the ProSA server was used to evaluate potential errors within the protein models. The analysis revealed negative z-values within a confidence zone, deviations that could be experimentally discerned through X-ray and NMR spectroscopy (Table.S3). In T. boeoticum, the predicted number of protein channels ranges from 1 to 7, with TbPYL5 having the highest number of channels (Table.S3). We predicted the protein binding pocket (Fig. 5b), whose surrounding amino acid residues determine its physicochemical properties, morphology, and function. Many functionally important residues are located in this pocket, including tyrosine (TYR), phenylalanine (PHE), asparagine (ASN), lysine (LYS), aspartic acid (ASP), glutamic acid (GLU), isoleucine (ILE), and leucine (LEU).
Collinearity analysis
Gene families can expand through tandem duplication and segmental duplication during evolution. To investigate these events, we defined a gene cluster as a chromosomal region spanning 200 kb or less and containing two or more genes30. The results showed that three gene clusters (TbPYL1 | TbPYL2, TbPYL7| TbPYL8, and TbPYL10 | TbPYL11) were identified from T. boeoticum (Fig. 2a). Simultaneously identifying duplicate of two fragments (TbPYL4| TbPYL12, and TbPYL5 | TbPYL13) (Fig. 6a). To further infer the phylogenetic mechanisms of the T. boeoticum PYL family, comparative synteny maps related to three different ploidy wheats (T. urartu, T. turgidum and T. aestivum) were constructed. The numbers of s collinear gene pairs with T. urartu, T. turgidum, and T. aestivum are 7, 33, and 49 (Fig. 6b-d, Table.S4), respectively. Seven of these genes have syntenic counterparts in all three species and 13 pairs of collinear PYL genes in Triticum turgidum and Triticum aestivum, with no collinearity genes of TbPYL6 and TbPYL11 (Fig. 6e, f).This indicates that most members of the PYL gene family in ancestral wheat varieties were relatively conserved throughout the entire evolutionary process and played an important role.
Interaction networks analysis of TbPYLs
To gain a better understanding of the biological functions and regulatory networks of TbPYLs, we performed a prediction of their protein-protein interactions (PPIs). Consistent with our expectations, the majority of proteins predicted to interact with TbPYLs are important components of the ABA signaling pathway, such as PP2C, ABI1 and DDA1 (Fig. 7a). Based on function annotation, these TbPYLs interacted proteins can be divided into phosphatase 2C family proteins (ABI1, ABI2, AHG1, AIP1, HAB1, HAB2, HAI3, PP2C and SAG113), DET1- and DDB1-associated protein (DDA1) and transcription factors (MYB44, MYB77 and MYC2). We also identified transcription factor binding sites for the PYL gene family, and found that ERF was the most abundant class of transcription factors, followed by MYB, bZIP, C2H2 and LBD, among others, indicating that multiple transcription factors are involved in the regulation of PYL genes (Fig. 7b). The ERF family is primarily involved in the ethylene signaling pathway, while the PYL family plays a key role in ABA signaling. It is widely recognized that ABA and ethylene interact antagonistically, influencing each other’s synthesis and their respective signaling transduction pathways31.
To investigate the potential association between ABA signaling pathways and miRNA regulation, we predicted putative miRNA targets of TbPYLs. Our analysis identified three miRNAs that may target four PYL genes (Fig. 7c). miR9666b-3p may target three PYL genes, suggesting its potential importance in ABA signaling.
Expression analysis of the PYL gene family
The expression profiles of TbPYLs in different tissues, including aerial, flag-leaf, spike, glume, grain and roots, were studied using RNA-seq data (Fig. 8a). The results indicate that these PYL genes exhibit certain tissue specificity. TbPYL1, TbPYL6, TbPYL7, TbPYL8 and TbPYL10 have higher expression levels in flag leaves than in other tissues. TbPYL2 shows high expression levels in most tissues, while TbPYL4, TbPYL5, TbPYL9, TbPYL12, TbPYL13, and TbPYL15 are mainly expressed at high levels in the glume. And only TbPYL14 is preferentially expressed in the spike. Additionally, consistent with previous studies, most PYL genes also exhibit certain levels of expression in grains. These results indicate that the TbPYLs play different roles in the growth and development of various tissues in T. boeoticum.
The expression patterns of wheat TbPYLs under cold stresses were also investigated based on RNA-seq data. TbPYL4, TbPYL6 and TbPYL14 were not expressed in the control and treatment groups, indicating that they were not involved in this process. Five PYL genes (TbPYL2, TbPYL5, TbPYL9 and TbPYL12) are induced by cold stress, with their expression levels showing varying degrees of increase compared to the control (Fig. 8b). TbPYL2 shows the most significant difference (TPM: 2.95–35.21). Based on the transcriptome data analysis mentioned above, four T. boeoticum PYL genes (TbPYL2, TbPYL5, TbPYL9 and TbPYL12) were selected for further validation. qPCR analysis was conducted to examine their expression levels after 24 hours of 4°C treatment in seedlings. Consistent with the transcriptome data, all four genes showed varying degrees of upregulation (Fig. 8c). These results indicate that TbPYLs play a certain role in response to cold stress.