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Article

Pan-Genome-Based Characterization of the PYL Transcription Factor Family in Populus

by
Xiaoli Han
1,2,3,
Chen Qiu
1,2,3,
Zhongshuai Gai
1,2,3,
Juntuan Zhai
1,2,3,
Jia Song
1,2,3,
Jianhao Sun
1,2,3,* and
Zhijun Li
1,2,3,*
1
Xinjiang Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin, Aral 843300, China
2
College of Life Science and Technology, Tarim University, Aral 843300, China
3
Desert Poplar Research Center, Tarim University, Aral 843300, China
*
Authors to whom correspondence should be addressed.
Plants 2025, 14(16), 2541; https://doi.org/10.3390/plants14162541
Submission received: 3 July 2025 / Revised: 6 August 2025 / Accepted: 6 August 2025 / Published: 15 August 2025
(This article belongs to the Special Issue Physiological and Molecular Mechanisms of Plant Resistance to Abiotic Stress)
Browse Figures
Versions Notes

Abstract

Abscisic acid (ABA) is a key phytohormone involved in regulating plant growth and responses to environmental stress. As receptors of ABA, pyrabactin resistance 1 (PYR)/PYR1-like (PYL) proteins play a central role in initiating ABA signal transduction. In this study, a total of 30 PopPYL genes were identified and classified into three sub-families (PYL I–III) in the pan-genome of 17 Populus species, through phylogenetic analysis. Among these subfamilies, the PYL I subfamily was the largest, comprising 21 members, whereas PYL III was the smallest, with only four members. To elucidate the evolutionary dynamics of these genes, we conducted synteny and Ka/Ks analyses. Results indicated that most PopPYL genes had undergone purifying selection (Ka/Ks < 1), while a few were subject to positive selection (Ka/Ks > 1). Promoter analysis revealed 258 cis-regulatory elements in the PYL genes of Populus euphratica (EUP) and Populus pruinosa (PRU), including 127 elements responsive to abiotic stress and 33 ABA-related elements. Furthermore, six structural variations (SVs) were detected in PYL_EUP genes and significantly influenced gene expression levels (p < 0.05). To further explore the functional roles of PYL genes, we analyzed tissue-specific expression profiles of 17 PYL_EUP genes under drought stress conditions. PYL6_EUP was predominantly expressed in roots, PYL17_EUP exhibited leaf-specific expression, and PYL1_EUP showed elevated expression in stems. These findings suggest that the drought response of PYL_EUP genes is tissue-specific. Overall, this study highlights the utility of pan-genomics in elucidating gene family evolution and suggests that PYL_EUP genes contribute to the regulation of drought stress responses in EUP, offering valuable genetic resources for functional characterization of PYL genes.

1. Introduction

Through evolutionary adaptation, plants have developed sophisticated regulatory networks to cope with abiotic stress [1,2,3,4], in which the phytohormone abscisic acid (ABA) plays a central role [5,6,7,8]. In plants, the ABA signaling cascade is initiated after ABA is perceived by pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/ABA receptors [9]. Under normal growth conditions, low ABA concentrations prevent PYR/PYL receptors from binding to PP2C phosphatases [10,11], thereby inhibiting SnRK2 kinase activity and holding the ABA signaling pathway in an inactive state [12]. During drought stress, elevated ABA levels induce PYL expression and promote its interaction with PP2C, relieving the inhibition of SnRK2 [13,14], activating downstream transcription factors and ion channels, and regulating stomatal closure and the expression of stress-responsive genes, ultimately enhancing plant drought resistance [15,16,17].
As members of the START protein superfamily, PYL proteins are characterized by a highly conserved START domain capable of binding hydrophobic ligands [18]. On the basis of this conserved gene sequence, 14 PYL genes containing the START domain have been identified in Arabidopsis thaliana and designated AtPYR1–AtPYR13. Phylogenetic analysis has classified these genes into three subfamilies [19,20]. Different subfamilies have been found to perform diverse functions in plant development and responses to abiotic stress. AtPYL5 and AtPYL9 have been identified as drought-resistant [21,22]. Under drought stress, AtPYL5 has been shown to enhance the photosynthetic rate [23]; AtPYR1, AtPYL2, AtPYL4, and AtPYL5 are involved in regulating stomatal and guard cell closure in response to CO2 [24]; and AtPYL8 and AtPYL9 play key roles in root growth and leaf senescence [25]. In addition, expression analysis of 11 ABA-related rate-limiting enzyme genes in different tissues of grapevine revealed that VvPYL1 exhibits tissue-specific expression, and the highest expression level has been observed in roots [26]. As a core regulator of ABA signaling, the function of PYL has been elucidated in many species, including rice (Oryza sativa) [27], tomato (Solanum lycopersicum) [7], soybean (Glycine max) [28], wheat (Triticum aestivum) [29], maize (Zea mays) [30], poplar (Populus) [31], rubber tree (Hevea brasiliensis) [32], strawberry (Fragaria × ananassa) [33], cotton (Gossypium hirsutum) [34], and castor bean (Ricinus communis) [35]. These studies demonstrate that the PYL gene family, as central components of the ABA signaling pathway, considerably enhances plant adaptation to abiotic stresses by regulating diverse physiological processes, making their functional characterization crucial for elucidating ABA signal transduction mechanisms [36].
With the advancement of plant pan-genomics, cross-species comparative genomic analysis has become feasible. Compared with conventional gene family identification approaches, pan-genomes overcome the limitation of single reference genomes by enabling the detection of gene family members that are absent from the reference genome but present in other genomes. This advancement provides new opportunities to investigate gene family conservation and divergence, as well as their roles in adaptive evolution [37]. Based on the pan-genome of 26 high-quality maize genomes, Sun et al. identified extensive presence–absence variations and structural variations (SVs), thereby laying the foundation for investigating the TPS gene family and its biological functions [38]. In addition, Wang et al. [39] constructed a genus-level super-pan-genome comprising 19 Populus genomes, identifying 142,202 cross-species SVs that intersected with numerous genes and significantly contributed to phenotypic and adaptive divergence. This study also provided comprehensive multi-omics resources associated with the Populus super-pan-genome. Although the structure and function of PYL genes have been extensively characterized in the model plant Arabidopsis thaliana and various crop species, a systematic study of the PYL gene family has not yet been conducted within the genus Populus of the family Salicaceae. Populus euphratica (EUP), known for its exceptional environmental adaptability and recognized as the most stress-resistant representative of the Turanga section of the genus Populus, is widely distributed across Central Asia, West Asia, and the arid regions of northwest China [40,41]. Its unique physiological and ecological adaptations enable remarkable survival under extreme drought and saline-alkali conditions, establishing it as an ideal model species for studying abiotic stress responses in woody plants [42]. In this study, we conducted a comprehensive identification and analysis of the PYL gene family across 17 species of the genus Populus, encompassing the sections Leucoides, Tacamahaca, Aigeiros, Turanga, and Leucoides. We conducted a comprehensive analysis of the gene structure, conserved motifs, phylogenetic relationships, promoter elements, SVs, and gene expression patterns of PYL proteins. In addition, tissue-specific expression profiles of PYL members in EUP were precisely quantified using qRT-PCR technology. These findings not only provide a timely foundation for functional studies of the PopPYL gene family but also significantly enhance our understanding of abiotic stress adaptation mechanisms in EUP, while offering valuable candidate genes for future genetic engineering and breeding programs in this species.

2. Results

2.1. Pan-Genome Distribution of the PopPYL Gene Family

Due to the limitations of a single reference genome in capturing the genetic diversity of a species, we collected 17 high-quality poplar genomes (Table 1) and conducted a pan-genome-level analysis of PYL genes to better understand their genetic variation. First, a comprehensive genome-wide comparative analysis of the PYL gene family was performed using BLASTP and HMM-based methods, resulting in the identification of 352 PYL genes in total. The number of PYL genes in each poplar genome ranged from 18 to 22 (Table S1). Orthologous genes across the 17 genomes were identified using OrthoFinder (v2.5.2; default parameters). The pan-gene IDs of the PYL gene family were extracted from the “Pan-genes.list” output file (Table S2), and genes sharing the same pan-gene ID across species were considered the same gene. Ultimately, 30 PYL pan-genes were obtained. Among these, 20 PopPYL genes were identified in Populus yunnanensis, Populus wuana (WUA), Populus trichocarpa (TRI), Populus simonii (SIM), and Populus lasiocarpa. Additionally, 18 PopPYL genes were detected in Populus tremula (TRE), Populus szechuanica (SZE), Populus pseudoglauca (PSE), Populus koreana, Populus davidiana, Populus alba var. pyramidalis (ALB), Populus adenopoda (ADE), Populus rotundifolia, and Populus qiongdaoensis (QIO). Notably, EUP (Table S3) and Populus pruinosa (PRU) exhibited the lowest number of PYL genes, with only 17 identified in each, whereas Populus deltoides (DEL) contained the highest number, with a total of 22 PYL genes.
Notably, QIO contained the unique genes Qdy09060.t1 (PYL3) and Qdy37849.t2 (PYL26), whereas ALB harbored three unique genes: pal_pou08344.t1 (PYL8), pal_pou07031.t1 (PYL21), and pal_pou33677.t1 (PYL22). Similarly, PRU possessed PprTF01G0764.1 (PYL23), ADE contained Poade06008.t2 (PYL24) and Poade13605.t2 (PYL25), SIM carried Posim17381.t2 (PYL27) and Posim35007.t2 (PYL28), TRE carried Potra2n8c17195.8 (PYL29), and WUA maintained Powua15969.t2 (PYL30). These species-specific genes may be associated with adaptive evolution in their respective species. Conversely, multiple PYL genes demonstrated high conservation across all genomes. Core PYL genes, including PYL1, PYL2, PYL4–7, and PYL9–17, were universally present in all 17 examined genomes. These conserved genes likely play essential roles in the fundamental physiological processes of Populus species and have remained evolutionarily stable.
Phylogenetic analysis of the Populus PYL protein family revealed three distinct subfamilies (I, II, and III), which exhibited significant divergence in their physicochemical properties, including amino acid length, molecular weight, isoelectric point (pI), instability index, and aliphatic index (Table 2). Subfamily I members displayed the greatest variability, with the amino acid length ranging markedly from 54 residues to 706 residues. PYL3 (706 amino acids) and PYL8 (615 amino acids) represented the largest proteins within this subfamily, whereas PYL18 (102–115 amino acids) was the smallest. This subfamily exhibited the broadest molecular weight distribution (9.7–77.3 kDa) and predominantly acidic isoelectric points (pI 4.4–10.2), and PYL21 (pI 8.44) was the sole alkaline exception. In contrast, subfamily II proteins exhibited a more conserved amino acid length (186–306 amino acids) and molecular weight (20.6–34.1 kDa), but were distinguished by their markedly alkaline isoelectric points (pI 7.1–9.2), a feature potentially related to their subcellular localization or functional specialization. The protein length of subfamily III members ranged from 169 amino acids (PYL20) to 201–259 amino acids (PYL7), with the molecular weight spanning 13.5–28.7 kDa. Notably, PYL7 exhibited the highest molecular weight within this subfamily (22.4–28.7 kDa). All members were characterized as acidic proteins (pI 4.93–7.01), and PYL9 showed the strongest acidity (pI 4.93–5.53). Hydrophilicity analysis (GRAVY index 0.29–0.52) confirmed their hydrophilic nature, among which PYL5 demonstrated the highest hydrophilicity.

2.2. Construction of the Phylogenetic Tree of PopPYL Gene Family Members

A phylogenetic tree was constructed based on the amino acid sequences of 14 AtPYL (Arabidopsis thaliana) and 30 PopPYL genes, revealing that the PopPYL proteins can be classified into three clades (PYL I, PYL II, and PYL III) following the established classification system for AtPYL, consistent with previous findings [51]. As shown in Figure 1, the PYL I subfamily was the most abundant, comprising 21 genes, followed by PYL II with 5 members, while PYL III was the smallest subfamily, containing only 4 members. This numerical disparity may reflect varying selective pressures and functional demands during the evolutionary diversification of these subfamilies. Within the PYL I subfamily, SZE uniquely possessed three copies of PYL13, whereas all other species retained only a single orthologous gene. Notably, PYL3 and PYL8 were detected exclusively in QIO and ALB, respectively, while PYL21–PYL30 exhibited single-species-specific distribution patterns. Additionally, EUP and PRU exhibited the loss of PYL15, although PRU uniquely harbored a distinct PYL23 gene. In the PYL II subfamily, PYL22 was identified as a species-specific gene in ALB, whereas PYL6, PYL10, and PYL11 were conserved as single-copy genes across all examined species. In contrast, PYL17 was absent in ALB, ADE, and QIO. For the PYL III subfamily, PYL7 and PYL9 were conserved as single copies across all 17 species, while PYL20 exhibited species specificity, being present only in WUA and PSE. The differential distribution of subfamily members across species suggests functional divergence within the PYL gene family, potentially linked to species-specific adaptations.

2.3. Chromosomal Location of PopPYL Genes in There Populus Species

Populus is a diploid species (2n = 18) with 18 chromosomes. To investigate the chromosomal distribution characteristics of PYL genes in poplar, we performed chromosomal localization analysis of 30 PopPYL genes (Figure 2). The results revealed both conserved features and species-specific variations in their distribution patterns. Overall, PYL genes were dispersed across multiple chromosomes without concentrated distribution on any single chromosome, reflecting genomic structural differences and evolutionary divergence among species. In the reference genome, we identified the following PYL genes: PYL1, PYL2, PYL4, PYL5, PYL6, PYL7, PYL9, PYL10, PYL11, PYL12, PYL13, PYL14, PYL16, and PYL17. Among these, PYL1, PYL2, PYL5, PYL6, PYL7, PYL9, PYL10, and PYL11 were conserved across all 17 Populus genomes, while PYL4, PYL12, PYL13, PYL14, PYL16, and PYL17 were present only in specific poplar species (Figure 2A). Additionally, 16 species-specific PYL genes were distributed across non-reference genomes (Figure 2B), with QIO, ALB, DEL, WUA, SIM, ADE, TRE, and PRU containing 2, 3, 3, 2, 2, 2, 1, and 1 gene, respectively. These genes compensate for the limitations of identifying gene family members using a single reference genome. In conclusion, the 30 homologous PopPYL genes collectively represent the PYL pan-genes across the 17 Populus species.

2.4. Gene Structure and Conserved Motif Analysis

Based on the evolutionary relationships between PopPYL and AtPYL genes, we analyzed the exon–intron structures of PYL6, PYL7, and PYL13, which are homologous to AtPYL5, AtPYR1, and AtPYL8, respectively, in 17 Populus species using TBtools software (v2.012). The results (Figure 3) revealed that PYL6 genes lacked introns in all 17 Populus species, while PYL13 genes consistently contained two introns. Members with identical or similar protein lengths shared conserved exon–intron structures, whereas genes with significant variation in protein length exhibited distinct gene architectures. For example, both PYL7_TRE and PYL7_PRU, which encode proteins of 255 and 259 amino acids, respectively, possessed two introns, whereas other Populus PYL7 genes encoding shorter proteins (201–207 amino acids) exhibited no detectable introns. This structural diversity may reflect species-specific physiological functions and environmental adaptations. Notably, PYL6, PYL7, and PYL13 genes all contained two exons across species, and homologous PYL genes maintained similar exon–intron patterns, further supporting their close evolutionary relationships and validating the current subfamily classification.
The conserved motifs of PYL6, PYL7, and PYL13 genes across 17 Populus species were further analyzed. PYL6 and PYL13 members exhibited highly similar motif types and arrangements, and all the members contained five conserved motifs (motif1–5), except PYL6_PSE, which harbored an additional motif (motif6; Figure 3). In contrast, PYL7 genes displayed considerable diversity in motif composition, and the number of motifs ranged from six to nine across species. Notably, motif1–5 was universally present in all PYL proteins, indicating a high degree of evolutionary conservation. Moreover, PopPYL genes with varying protein lengths and gene structures showed distinct motif patterns. For example, within the PYL7 subfamily, PYL7_TRE and PYL7_PRU exhibited longer protein lengths, divergent gene structures, and a greater number of conserved motifs compared to other Populus species. These findings suggest that PopPYL members within the same subfamily share conserved motif characteristics, supporting functional conservation, while the presence of unique motifs may confer specialized biological functions.

2.5. Synteny Analysis of PopPYL Genes

Genome-wide synteny analysis of PYL6, PYL7, PYL9, and PYL13 genes among four Populus species (EUP, TRI, PRU, and ADE) identified 61 syntenic gene pairs. Specifically, 14 homologous gene pairs were observed between TRI and ADE, while 11 pairs were detected between EUP and TRI, as well as between TRI and PRU. Additionally, 10 homologous pairs were identified between EUP and ADE, 9 between PRU and ADE, and 6 between EUP and PRU (Figure 4A). These findings suggest that the evolution and expansion of these genes primarily resulted from whole-genome duplication events.
Further Ka/Ks analysis of the PYL gene family across 17 Populus species revealed that most gene pairs exhibited Ka/Ks ratios significantly less than 1, indicating strong purifying selection during evolution (Figure 4B). This conservation of key functional domains is likely associated with their essential roles in plant stress adaptation. However, certain gene pairs, including PYL4, PYL5, PYL6, PYL7, PYL9, PYL10, PYL11, PYL12, PYL14, PYL15, and PYL19, displayed Ka/Ks values greater than 1, suggesting potential positive selection in some Populus lineages (Figure 4C). Notably, PYL4, PYL6, PYL7, PYL9, PYL14, and PYL15 frequently exhibited Ka/Ks > 1 across multiple species, implying strong positive selection pressure during Populus evolution. In contrast, PYL5, PYL10, PYL11, and PYL12 appeared to undergo positive selection only in specific species.

2.6. Promoter Analysis of PopPYL Genes

To elucidate the transcriptional regulatory mechanisms of PYL_EUP and PYL_PRU, a comparative analysis and PlantCARE-based screening of their promoter regions (2 kb upstream of the start codon) was performed (Figure 5A). A total of 258 cis-regulatory elements were identified and classified into five functional categories: plant growth and development (29 elements), abiotic stress response (127 elements), hormone signaling (39 elements), ABA-responsive elements (33 elements), and light response (30 elements). Notably, the AREB elements (ABA-responsive) were ubiquitously present, the highest abundance (five elements) was observed in PYL17_PRU, and only one was detected in PYL7_EUP/PRU (Figure 5B). Among abiotic stress-responsive elements, MYB elements associated with environmental adaptation exhibited significant variation in distribution. PYL17_EUP/PRU contained the fewest (2 elements), whereas PYL9_EUP/PRU exhibited the highest number (11 elements). Additionally, long terminal repeat (LTR) retrotransposon sequences were identified in PYL7/9/13/17_EUP. Hormone-responsive elements showed distinct patterns: auxin response elements (AREs) were widely distributed, with maximum enrichment in PYL13_EUP (ten elements) and a minimum of one element in PYL7_EUP (exclusively present in P. euphratica). JA/MeJA-responsive elements (TGACG motif and CGTCA motif) were restricted to PYL17_EUP/PRU and PYL1_PRU (≥1 element each), while SA-responsive (TCA element) and gibberellin-responsive elements (GARE motif) were sparsely distributed. Growth- and development-related CAT-box elements exhibited a broad distribution, peaking in PYL17_EUP/PRU (five elements) and with the lowest abundance in PYL1_EUP (one element, specific to EUP). Light-responsive G-box elements were found in all genes except PYL13_PRU, with maximum enrichment in PYL1_EUP/PRU (five elements). P-box elements were exclusively detected in PYL7_EUP/PRU and PYL17_EUP/PRU.

2.7. Structural Variation Analysis of PYL_EUP Genes

Structural variations (SVs), commonly defined as DNA sequence alterations exceeding 50 base pairs (bp) in length, encompass a range of mutation types including insertions (INS), deletions (DEL), inversions, translocations, and copy number variations. These genomic alterations can directly affect protein function or gene expression by modifying coding sequences, gene copy numbers, or regulatory regions, thereby contributing to species evolution and phenotypic diversity. In the present study, a pan-genome was constructed using P. euphratica (EUP) as the reference genome, and SV analysis was conducted across 17 Populus species. Within the PYL gene family of P. euphratica, six structural variation sites were identified, primarily located in upstream, downstream, and intronic regions, comprising two insertions and four deletions (Figure 6). Specifically, one insertion (562 bp) was located upstream of Peu06G022430 on LG06, and another (32 bp) was found upstream of Peu10G016340 on LG10, both potentially introducing novel cis-regulatory elements. Meanwhile, a 258 bp deletion occurred 459 bp downstream of Peu01G012370 on LG01, and a 268 bp deletion occurred 271 bp downstream of Peu02G014980 on LG02, which may attenuate transcriptional regulation of these genes. In addition, intronic regions of Peu15G001800 and Peu02G014980 harbored a 1 bp insertion and a 725 bp deletion, respectively. These variations can disrupt mRNA splicing efficiency and result in aberrant transcripts. To evaluate the functional impact of these SVs, we employed Mann–Whitney U tests to compare expression levels between PYL genes with and without SVs. The results revealed statistically significant differences (p < 0.05), suggesting that structural variations substantially influenced the expression patterns of PYL_EUP genes. Collectively, these findings provided novel molecular insights into the evolutionary dynamics and functional diversification of the PYL gene family in Populus.

2.8. Expression Analysis of the PYL_EUP Gene Family

Abscisic acid (ABA), a key phytohormone involved in regulating plant growth and stress responses, initiates its signaling pathway through recognition by the Pyrabactin Resistance 1-like (PYR/PYL) receptor family. Based on RNA-seq data analysis of P. euphratica under drought stress (Table S4), 16 PYL_EUP genes, excluding PYL16_EUP, exhibited differential expression patterns across root, stem, and leaf tissues (Figure 7A). Notably, PYL1/2/6/9/11/13_EUP were predominantly expressed in roots, suggesting their involvement in root-cap ABA signaling transduction. In contrast, PYL5/7/12/17_EUP showed leaf-specific upregulation, indicating potential roles in stomatal closure or leaf developmental processes. Meanwhile, the stem-preferential expression of PYL1/4/10/14_EUP implied a regulatory function in coordinating whole-plant drought responses, possibly by mediating ABA long-distance transport through phloem or xylem vascular tissues. This organ-specific divergence in expression clearly demonstrated the functional specialization of PYL_EUP family members across different tissues of P. euphratica, thereby optimizing ABA-mediated adaptation to drought stress.
To elucidate the potential roles of PYL_EUP genes in response to drought stress, their expression patterns were analyzed across different seedling tissues subjected to drought treatment. Heatmap analysis revealed distinct tissue-specific expression profiles among PYL family members under drought conditions (Figure 7B–D). These patterns suggest a functional differentiation of PYL_EUP genes in mediating ABA signaling and stress adaptation in a tissue-dependent manner. In leaves, PYL1_EUP, PYL7_EUP, and PYL13_EUP were upregulated under drought conditions, whereas PYL2/4/5/6/10/11/12/14/17_EUP exhibited downregulated expression (Figure 7B). In stems, only PYL1_EUP showed increased expression during drought stress, while all other PYL_EUP genes were downregulated (Figure 7C). In roots, PYL1_EUP, PYL4_EUP, PYL12_EUP, and PYL13_EUP were upregulated, and the remaining genes showed reduced expression levels (Figure 7D). Notably, PYL1_EUP demonstrated consistent upregulation in roots, stems, and leaves, highlighting its potential central role in the core regulatory network of the drought stress response in EUP. These results suggest that different PYL_EUP genes may contribute to drought adaptation through tissue-specific and functionally distinct regulatory mechanisms.

2.9. Verification of PYL Gene Expression Patterns in Different Tissues by RT-qPCR

To further elucidate the functional characteristics of the PYL_EUP gene family under drought stress, we performed a comparative expression analysis of six PYL_EUP genes across different plant tissues using quantitative real-time PCR (qRT-PCR). The results revealed significant tissue-specific expression differences among the selected PYL genes (Figure 8). Notably, PYL7_EUP, PYL9_EUP, and PYL17_EUP exhibited relatively higher expression levels in seedling leaves. In particular, PYL7_EUP showed peak expression in leaves and moderate expression in stems, whereas PYL17_EUP, although little expressed overall, displayed distinct preferential expression in leaf tissue. Conversely, PYL13_EUP was predominantly expressed in seedling stems. Additionally, PYL1_EUP, PYL6_EUP, and PYL13_EUP were primarily expressed in roots, and PYL1_EUP exhibited markedly higher expression in roots than in other tissues. PYL6_EUP demonstrated a graded expression pattern across organs, while PYL13_EUP showed its highest expression in root tissue. Validation of gene expression patterns demonstrated a strong correlation between qRT-PCR (2–ΔΔCt) and RNA-seq (FPKM) data for most genes (PYL1, PYL6, PYL7, PYL13, and PYL17). These findings underscore the functional diversification of PYL family members during drought adaptation and provide valuable molecular insights for future mechanistic studies.

3. Discussion

In this study, a high-quality pan-genome was constructed based on 17 Populus species, overcoming the limitations associated with relying on a single reference genome and enabling the comprehensive identification of full-spectrum genetic variations, including non-reference genes. By aligning 14 Arabidopsis AtPYL protein sequences, a total of 30 Populus PYL genes were identified in the pan-genome, comprising 17 reference-based genes and 13 non-reference genes. Among these, DEL possessed the highest number of PYL genes (22), while EUP and PRU exhibited the lowest gene counts (17). This variation in gene number may reflect divergent evolutionary trajectories among Populus species, and certain lineages underwent multiple gene duplication events [39] that contributed to the expansion of the PYL gene family. However, the duplicated genes have been subject to species-specific selective pressures, resulting in different evolutionary outcomes. Some PYL genes were conserved across most species due to essential functional roles, while others experienced functional divergence or were lost entirely in certain lineages. For instance, the eight PopPYL genes present in all species likely play central and conserved roles in fundamental biological processes, such as plant hormone signaling, cell growth, and development. In contrast, the 12 PopPYL genes unique to individual species might contribute to species-specific ecological adaptations or physiological functions, potentially aiding in responses to environmental stresses or specialized growth and developmental processes.
This study investigated the structural characteristics of PYL6, PYL7, and PYL13 genes across 17 Populus species, revealing potential functional divergence within this gene family. The results showed that PYL13 consistently contained two introns in all species, maintaining a highly conserved intron–exon structure across Populus. In contrast, PYL7 exhibited substantial SV. Only PYL7_TRE and PYL7_PRU possessed two introns, whereas other members were intronless. This divergence may reflect functional differentiation following gene duplication, paralleling observations in Arabidopsis, where AtPYL1–AtPYL8 typically contain 1–2 introns, while AtPYL9–AtPYL13 are predominantly intronless, suggesting distinct evolutionary trajectories among subfamilies [52]. Motif analysis revealed that motifs 1–5 were universally present across all PYL proteins, likely corresponding to core functional domains of START-like ABA receptors, consistent with conserved ABA-binding sites reported in Arabidopsis PYL proteins [20]. However, PYL7 members displayed notable diversity in motif composition (6–9 motifs), particularly due to the presence of unique additional motifs in PYL7_TRE and PYL7_PRU, which were associated with their extended protein length. Similar patterns were observed in the tomato SlPYL family, where C-terminal extensions conferred regulatory specificity to certain members [53]. These variations may reflect adaptive responses to drought or cold stress. Furthermore, significant differences in protein length and motif organization among PYL7 members (e.g., PYL7_TRE with 259 amino acids versus 201–207 in others) suggest potential subfunctionalization or neofunctionalization. For instance, in maize, ZmPYL10 acquired distinct ABA sensitivity through the insertion of a specific sequence [54].
Gene family expansion in plant evolution is primarily driven by whole-genome duplication (WGD) and tandem duplication [55]. Our multiple synteny analysis of four core Populus PYL genes revealed extensive syntenic relationships across species, consistent with the predominant mechanisms of plant gene family expansion. Previous studies have shown that the genus Populus underwent a unique WGD event, namely, the Salicoid duplication, approximately 60 million years ago, which contributed to the expansion of numerous gene families, including those involved in ABA signaling pathways [56]. Selection pressure analysis revealed that the majority of PYL gene pairs exhibited Ka/Ks ratios significantly less than 1 (p < 0.01), indicating strong purifying selection during evolution. This likely preserved their conserved functions in ABA perception and signal transduction. These findings are consistent with previous studies on the rice OsPYL family, in which the START-like domain of ABA receptor proteins demonstrated high conservation [57]. However, PYL4, PYL6, PYL7, PYL9, PYL14, and PYL15 frequently showed Ka/Ks > 1 across multiple Populus species, suggesting that these genes may confer enhanced adaptive advantages under specific environmental stresses, such as drought or salinity. Similarly, the positive selection signals observed in PYL6 and PYL9 may reflect species-specific adaptation strategies to abiotic stresses, analogous to the drought tolerance conferred by positive selection in maize ZmPYL10 [54].
Insertion and deletion (InDel) variations in genomes can directly alter gene coding sequences, resulting in amino acid changes or frameshift mutations that ultimately affect protein function [58]. For example, in peanut (Arachis hypogaea), an insertion variant in the 3′-UTR of AhCKX6 reduced gene expression and increased active cytokinin levels, thereby promoting grain enlargement [59]. Similarly, a 358 bp insertion in the promoter region of maize bZIP68 significantly upregulated its expression [60]. Structural variations (SVs) also play crucial roles in plant stress adaptation and evolutionary processes. In rice, for instance, long SVs are significantly enriched in promoter regions, and strong colocalization was observed between SVs and stress-responsive genes, suggesting that SVs contribute to the pleiotropic expression patterns commonly observed in stress-related genes [61]. In our investigation of the PYL gene family in EUP, we identified six SVs (two insertions and four deletions) predominantly located in regulatory regions. These included 1 bp and 32 bp insertions upstream, and 258 bp and 268 bp deletions downstream of PYL genes. These SVs may influence gene expression by modifying regulatory sequences or interfering with splicing, thus modulating ABA signaling pathways and environmental adaptation. Notably, SVs within intronic regions (a 1 bp insertion and a 725 bp deletion) can alter the spatial configuration of splice donor or acceptor sites, potentially increasing alternative splicing events. As demonstrated in rice Ptr gene studies, atypical gene structures induced by SVs (including variations in exon number or protein truncation) may still retain essential biological functions [62].
cis-acting elements in plant PYL genes play pivotal roles in environmental stress responses and signaling pathways that govern plant growth and development [36]. These regulatory elements can be classified into three major categories: hormone-responsive elements (e.g., ABRE) [63], growth-related elements (e.g., CAT-box) [64], and stress-responsive elements (e.g., MBS) [12]. Our systematic analysis of promoter regions (2 kb upstream) in PYL gene families from EUP and PRU identified 64 distinct cis-acting elements, encompassing light-responsive, abiotic stress-related, and hormone-responsive elements. As crucial components of ABA signaling, PYL gene promoters are particularly enriched with AREB elements, and PYL17_PRU exhibits the highest abundance. These findings suggest PYL17_PRU’s potential significance in ABA-mediated stress responses [65]. Notably, ARE elements involved in phytohormone responses exhibit substantial distribution variation, being exclusively present in EUP, with maximal enrichment in PYL13_EUP. Among abiotic stress-related elements, MYB transcription factors, which regulate plant cell morphogenesis [66], developmental processes [67], and stress adaptation [68], showed marked distribution variation. PYL9_EUP/PRU demonstrated the highest density, indicating its enhanced environmental stress sensitivity. Furthermore, JA/MeJA-responsive elements were uniquely distributed in PYL17_EUP/PRU and PYL1_PRU, implying their potential involvement in jasmonate signaling-mediated stress resistance. The distinct distribution patterns of these cis-elements reveal the sophisticated transcriptional regulatory mechanisms underlying the multifunctional roles of PYL genes in plant development, hormone signaling, and stress adaptation.
Gene expression patterns are closely related to biological functions. This study systematically analyzed the tissue-specific expression and drought stress response characteristics of the PYL_EUP gene family in EUP, providing insights into its physiological roles and regulatory mechanisms. Previous studies have demonstrated that plant PYL gene family members generally exhibit tissue-specific expression patterns. In soybean seeds, most PYL genes showed significantly higher expression levels than in other tissues [28]. During oilseed germination, PYL genes displayed high transcriptional abundance [69]. In rubber tree latex, PYL gene transcripts were particularly abundant [32]. Additionally, PYL genes exhibited predominant expression in Chinese cabbage callus [70]. Our research revealed distinct differential expression patterns of 17 PYL_EUP genes (excluding PYL16_EUP) in EUP across different tissues: PYL1/2/6/9/11/13_EUP was significantly enriched in roots, PYL5/7/12/17_EUP showed specifically high expression levels in leaves, and PYL1/4/10/14_EUP exhibiting predominant expression in stems.
Phylogenetic analysis of PYL_EUP genes and their Arabidopsis thaliana homologs revealed that PYL1_EUP, PYL6_EUP, PYL7_EUP, PYL9_EUP, PYL13_EUP, and PYL17_EUP were orthologous to AtPYL9, AtPYL5, AtPYR1, AtPYL1, AtPYL8, and AtPYL4, respectively [51]. Functional studies have demonstrated that AtPYL9 overexpression accelerated senescence and death in older leaves while promoting summer dormancy-like responses in young tissues under severe water deficit [71]; AtPYL4 overexpression significantly enhanced drought resistance [36]; AtPYL8 regulated lateral root growth, with its mutation causing ABA insensitivity in roots during stress recovery [25]; and AtPYL5 plays essential roles in stomatal responses to ABA and environmental signals by integrating various stimuli to balance water loss and CO2 uptake [24]. Our transcriptomic and qPCR analyses revealed that PYL_EUP genes exhibited tissue-specific induction under drought stress. Specifically, PYL1_EUP was upregulated in roots, stems, and leaves, whereas PYL7_EUP and PYL13_EUP showed leaf-specific induction. In contrast, PYL4/12/13_EUP exhibited root-specific upregulation. These results suggested that PYL_EUP genes participated in the drought response of P. euphratica through tissue-specific regulatory mechanisms. Notably, PYL1_EUP appeared to function as a master regulator coordinating whole-plant responses, while other members executed tissue-specific functions, collectively establishing a robust drought response system.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

This experiment selected P. euphratica seeds from the Awati County P. euphratica Forest in Xinjiang (located at the northwestern edge of the Tarim Basin, characterized by a warm-temperate extreme continental arid desert climate). Mature capsules were collected in September 2022, followed by natural drying under indoor conditions, separation of pappus from seeds, and storage in sealed brown bottles at 4 °C for later use. The seeds were uniformly sown in nutrient soil trays and cultivated in a greenhouse at 30 °C, 50% humidity, and under a 16 h light/8 h dark photoperiod for two months. Subsequently, the seedlings were transplanted to larger trays, with one seedling retained per tray for continued growth. A completely randomized design was adopted, using one-year-old P. euphratica seedlings as experimental material for drought treatment. Sixty healthy seedlings with uniform growth and no pests or diseases were selected and evenly divided into two groups: one subjected to drought stress and the other as a well-watered control group, with three biological replicates per group. After treatment, both groups were maintained under identical growth conditions. When leaf wilting occurred and the soil moisture content reached 21.5%, root, stem, and leaf tissues from seedlings of both groups were collected on the 13th day, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent transcriptomic analysis.

4.2. Data Acquisition

The genomic sequences, genome annotation files, protein sequences, and bioinformatics data of PSE, WUA, SZE, YUN, KOR, SIM, LAS, DAV, ROT, ALB, QIO, and ADE were obtained from the China National Center for Bioinformation (CNCB) (https://bigd.big.ac.cn/gwh (accessed on 8 April 2025)). The genomic data of TRI and DEL were sourced from https://phytozome-next.jgi.doe.gov/ (accessed on 8 April 2025). The genomic data of TRE were obtained from http://popgenie.org (accessed on 8 April 2025). The genomic data of PRU were sourced from Figshare (https://figshare.com/articles/online_resource/Pprgenome_fa/20705107/2 (accessed on 8 April 2025)). The telomere-to-telomere (T2T) complete genome assembly of Populus euphratica used in this study was independently generated by our research group and has not been publicly released. The genomic and proteomic data of Arabidopsis thaliana PYL family members were retrieved from the TAIR database (https://www.arabidopsis.org/ (accessed on 10 April 2025)).

4.3. Identification of PopPYL Genes in Populus Species

In this study, two complementary approaches were employed to systematically identify PYL homologs. First, the BLASTP algorithm was utilized to search the protein databases of Arabidopsis thaliana and 17 Populus species, using 13 Arabidopsis PYL protein sequences as queries. The E-value threshold was set to 1e−5 to ensure stringent homology detection. Second, an HMM-based search was conducted by scanning the same protein datasets against the PYL-specific domain model (PF10604.hmm, retrieved from the Pfam database, https://www.ebi.ac.uk/interpro/ (accessed on 11 April 2025)) [72]. The union of candidate genes obtained from both methods was then subjected to further validation. SMART v8.0 (http://smart.embl-heidelberg.de/ (accessed on 11 April 2025)) and CD-Search v3.20 (https://www.ncbi.nlm.nih.gov/Structure/ (accessed on 11 April 2025)) were used to confirm the presence of complete PYL conserved domains, eliminating false-positive hits. The final high-confidence gene list was compiled, and TBtools was employed for batch calculation of molecular weights and isoelectric points (pI) of the identified PYL proteins.

4.4. Phylogenetic Analysis of PopPYL Family Protein Sequences

Multiple sequence alignment of PopPYL family protein amino acid sequences was conducted using the MUSCLE algorithm implemented in MEGA 7.0 software. Based on the alignment results, a phylogenetic tree was reconstructed through the Neighbor-Joining (NJ) method with 1000 bootstrap replicates to evaluate nodal support reliability. The final phylogenetic tree (Newick format: *.nwk) was subsequently visualized and annotated using the iTOL v6 online platform (http://itol.embl.de/ (accessed on 12 April 2025)).

4.5. Motif Analysis and Gene Structure

The conserved motifs in PopPYL proteins were predicted using the MEME Suite v 5.5.5 (https://memesuite.org/meme/tools/meme (accessed on 14 April 2025)) with parameters set to 10 motifs per sequence and an E-value cutoff < 1 × 10−5 [73]. Functional domain annotation was performed using NCBI’s Batch CD-Search v3.20 tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi (accessed on 14 April 2025)) with default threshold settings. The predicted motifs and annotated domains were integrated and visualized using TBtools software (v2.309) to generate comprehensive schematic representations.

4.6. Gene Duplication Analysis and Ka/Ks Calculation

Gene Duplication Analysis: Whole-genome synteny analysis of PYL genes was conducted among EUP, PRU, TRI, and ADE lineages using TBtools software. Homologous gene pairs were identified through whole-genome alignment with the “MCScanX” module in TBtools, based on species-specific genome annotation files and genomic sequences.
Ka/Ks Analysis: Evolutionary selection pressure was assessed by calculating Ka and Ks values with Ka/Ks ratios using Ka/Ks_Calculator v2.0 [74]. Generally, Ka/Ks = 1 indicates neutral mutation, Ka/Ks > 1 suggests positive selection, and Ka/Ks < 1 represents purifying selection.

4.7. SVs and Gene Expression Analysis of PYL_EUP

This study constructed a pan-genome of 17 Populus species using the P. euphratica (EUP) reference genome and performed structural variant (SV) analysis, generating a novel SV dataset (17genome.sv.vcf). Functional annotation of SVs was conducted with ANNOVAR (2018Apr16) to extract key VCF features including chromosomal coordinates, variant boundaries, and reference/alternative alleles. Through custom scripts, we characterized SV distributions in genic regions (coding sequences, introns) and flanking regulatory regions (±2 kb). SVs associated with the PYL_EUP gene family were filtered from the annotated results. Gene expression profiles across all 17 Populus samples were obtained from the PSIR database (http://www.populus-superpangenome.com/ (accessed on 20 April 2025)). The Mann–Whitney U test was systematically employed to assess the association between SV presence/absence patterns and PYL gene expression levels. PYL genes with p-values < 0.05 were classified as showing SV-associated expression alterations.

4.8. Analysis of Promoter Regions of PopPYL Genes

The 2000 bp upstream sequences from the translation start sites of PYL_EUP and PYL_PRU genes were extracted using TBtools (v2.309). These promoter sequences were then analyzed in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 23 April 2025)) to predict putative cis-regulatory elements. The identified cis-acting elements were visualized using TBtools (v2.309) [75].

4.9. Analysis of Gene Expression Profiles

Differential gene expression analysis between sample groups was conducted using DESeq2 to identify differentially expressed genes (DEGs) under two biological conditions [76]. The obtained P-values were adjusted for multiple testing using the Benjamini–Hochberg method to control the false discovery rate (FDR). DEGs were filtered with thresholds of |log2FoldChange| ≥ 1 and FDR < 0.05. Expression patterns of PYL_EUP and PYL_PRU genes were visualized through heatmaps generated with TBtools software (v2.309).

4.10. qRT-PCR Validation

To further validate the accuracy of the sequencing data, we selected six significantly differentially expressed genes (DEGs) in different tissues of P. euphratica for qRT-PCR verification. Gene-specific primers for qRT-PCR were designed using the NCBI Primer-BLAST tool v6.25 (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 25 April 2025)) (Table S5). Total RNA was extracted using the Total RNA Extractor (Trizol) Plant RNA Kit (B511311, Sangon Biotech (Shanghai, China)). Genomic DNA contamination was evaluated by 1.5% agarose gel electrophoresis, and RNA concentration and OD values were determined. First-strand cDNA was synthesized using Maxima Reverse Transcriptase (EP0743, Thermo Fisher Scientific). qPCR amplification was performed on an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) in 20 μL reaction volumes containing 10 μL SGExcel FastSYBR Mixtures, 0.4 μL each of forward and reverse primers (10 μM), 7.2 μL RNase-Free ddH2O, and 2 μL cDNA template. The thermal cycling conditions consisted of initial denaturation at 95 °C for 3 min, followed by 45 cycles of 95 °C for 15 sec and 60 °C for 45 sec (annealing/extension). Three biological and technical replicates were performed for each sample.

5. Conclusions

A pan-genomic analysis of 17 Populus species identified 30 PopPYL pan-genes exhibiting significant copy number variation, representing 325 distinct PYL gene homologues. These findings indicate frequent gene duplication and loss events during Populus evolution. Phylogenetic classification divided PopPYL into three clades (I–III), where clade III was the most expanded (21 members) and clade I the most conserved (four members). Species-specific distribution patterns were observed for PYL3 and PYL8, while the absence of PYL15 in EUP and PRU, and the unique acquisition of PYL23, reflected functional remodeling during adaptive evolution. Gene structure and conserved motif analyses revealed similar exon–intron architectures and motif compositions within subfamilies, although distinctive SVs in PYL7_TRE/PRU indicated potential neofunctionalization. Synteny and selection pressure analyses identified 61 homologous gene pairs resulting from whole-genome duplication events, and PYL4/6/7/9 showed positive selection (Ka/Ks > 1) across multiple species, consistent with their roles in drought response. Promoter analysis revealed complex regulatory networks, featuring abundant MYB elements in PYL9_EUP/PRU, enriched ARE elements in PYL13_EUP, and the highest ABRE content in PYL17_PRU, providing a basis for tissue-specific expression. SV analysis detected six SV loci significantly affecting PYL gene expression patterns (p < 0.05). Expression profiling confirmed distinct functional specialization: root-enriched PYL1/6/13_EUP likely regulated root-cap ABA signaling; leaf-specific PYL7/17_EUP might participate in stomatal regulation; while stem-predominant PYL1/4/10_EUP potentially coordinated long-distance ABA transport. Crucially, PYL1_EUP showed drought-induced upregulation in roots, stems, and leaves, and PYL1 and PYL13 exhibited high expression levels across detection methods, demonstrating their roles as core regulatory nodes in poplar drought resistance. These findings advance our understanding of the biological functions of PYL genes and provide a foundation for breeding stress-resistant poplar varieties.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/plants14162541/s1, Table S1. Identification of PYL genes in the 17 Populors; Table S2. Pan-gene list of the 17 Populors; Table S3. PYL gene family of Populus euphratica (EUP); Table S4. RNA-seq data of PYL_EUP; Table S5. Primer sequences.

Author Contributions

Conceptualization, Z.L. and J.S. (Jianhao Sun); methodology, Z.G. and J.S. (Jianhao Sun); formal analysis, X.H. and C.Q.; investigation, X.H. and J.Z.; data curation, X.H. and J.S. (Jia Song); writing—original draft preparation, X.H.; writing—review and editing, Z.L. and J.S. (Jianhao Sun); visualization, X.H.; project administration, Z.L.; funding acquisition, Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the General Program of the National Natural Science Foundation of China (32371838), the second batch of the ‘Tianshan’ Support Program—Technology Innovation Team (2023TSYCTD0019), the Guidance Program for Science & Technology Projects of XPCC (2023ZD091), and the Start-up Research Fund for Doctoral Scholars from the President’s Fund of Tarim University (TDZKBS202532).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Acknowledgments

We sincerely acknowledge the State Key Laboratory of Tarim Basin Biological Resources Protection and Utilization of Xinjiang Production and Construction Corps for providing the advanced research platform and technical support for this study. All computations in this paper were performed on the bioinformatics computing platform of Tarim University.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Shinozaki, K.; Yamaguchi-Shinozaki, K. Gene networks involved in drought stress response and tolerance. J. Exp. Bot. 2007, 58, 221–227. [Google Scholar] [CrossRef]
  2. Cramer, G.R.; Urano, K.; Delrot, S.; Pezzotti, M.; Shinozaki, K. Effects of abiotic stress on plants: A systems biology perspective. BMC Plant Biol. 2011, 11, 163. [Google Scholar] [CrossRef]
  3. Santos, A.P.; Serra, T.; Figueiredo, D.D.; Barros, P.; Lourenço, T.; Chander, S.; Oliveira, M.M.; Saibo, N.J. Transcription regulation of abiotic stress responses in rice: A combined action of transcription factors and epigenetic mechanisms. Omics J. Integr. Biol. 2011, 15, 839–857. [Google Scholar] [CrossRef]
  4. Friedel, S.; Usadel, B.; von Wirén, N.; Sreenivasulu, N. Reverse engineering: A key component of systems biology to unravel global abiotic stress cross-talk. Front. Plant Sci. 2012, 3, 294. [Google Scholar] [CrossRef]
  5. Cutler, S.R.; Rodriguez, P.L.; Finkelstein, R.R.; Abrams, S.R. Abscisic acid: Emergence of a core signaling network. Annu. Rev. Plant Biol. 2010, 61, 651–679. [Google Scholar] [CrossRef] [PubMed]
  6. Fujii, H.; Zhu, J.K. Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc. Natl. Acad. Sci. USA 2009, 106, 8380–8385. [Google Scholar] [CrossRef] [PubMed]
  7. Gonzalez-Guzman, M.; Pizzio, G.A.; Antoni, R.; Vera-Sirera, F.; Merilo, E.; Bassel, G.W.; Fernández, M.A.; Holdsworth, M.J.; Perez-Amador, M.A.; Kollist, H.; et al. Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid. Plant Cell 2012, 24, 2483–2496. [Google Scholar] [CrossRef]
  8. Munemasa, S.; Hauser, F.; Park, J.; Waadt, R.; Brandt, B.; Schroeder, J.I. Mechanisms of abscisic acid-mediated control of stomatal aperture. Curr. Opin. Plant Biol. 2015, 28, 154–162. [Google Scholar] [CrossRef]
  9. Belda-Palazon, B.; Gonzalez-Garcia, M.P.; Lozano-Juste, J.; Coego, A.; Antoni, R.; Julian, J.; Peirats-Llobet, M.; Rodriguez, L.; Berbel, A.; Dietrich, D.; et al. PYL8 mediates ABA perception in the root through non-cell-autonomous and ligand-stabilization-based mechanisms. Proc. Natl. Acad. Sci. USA 2018, 115, E11857–E11863. [Google Scholar] [CrossRef]
  10. Schweighofer, A.; Hirt, H.; Meskiene, I. Plant PP2C phosphatases: Emerging functions in stress signaling. Trends Plant Sci. 2004, 9, 236–243. [Google Scholar] [CrossRef] [PubMed]
  11. Saavedra, X.; Modrego, A.; Rodríguez, D.; González-García, M.P.; Sanz, L.; Nicolás, G.; Lorenzo, O. The nuclear interactor PYL8/RCAR3 of Fagus sylvatica FsPP2C1 is a positive regulator of abscisic acid signaling in seeds and stress. Plant Physiol. 2010, 152, 133–150. [Google Scholar] [CrossRef] [PubMed]
  12. Zhou, J.; An, F.; Sun, Y.; Guo, R.; Pan, L.; Wan, T.; Hao, Y.; Cai, Y. Genome-wide identification of the ABA receptor PYL gene family and expression analysis in Prunus avium L. Sci. Hortic. 2023, 313, 111919. [Google Scholar] [CrossRef]
  13. Yuan, X.; Yin, P.; Hao, Q.; Yan, C.; Wang, J.; Yan, N. Single Amino Acid Alteration between Valine and Isoleucine Determines the Distinct Pyrabactin Selectivity by PYL1 and PYL2. J. Biol. Chem. 2010, 285, 28953–28958. [Google Scholar] [CrossRef]
  14. Fujita, Y.; Nakashima, K.; Yoshida, T.; Katagiri, T.; Kidokoro, S.; Kanamori, N.; Umezawa, T.; Fujita, M.; Maruyama, K.; Ishiyama, K.; et al. Three SnRK2 protein kinases are the main positive regulators of abscisic acid signaling in response to water stress in Arabidopsis. Plant Cell Physiol. 2009, 50, 2123–2132. [Google Scholar] [CrossRef]
  15. Choi, H.-i.; Hong, J.-h.; Ha, J.-o.; Kang, J.-y.; Kim, S.Y. ABFs, a Family of ABA-responsive Element Binding Factors. J. Biol. Chem. 2000, 275, 1723–1730. [Google Scholar] [CrossRef]
  16. Yang, W.; Zhang, W.; Wang, X. Post-translational control of ABA signalling: The roles of protein phosphorylation and ubiquitination. Plant Biotechnology Journal. 2017, 15, 4–14. [Google Scholar] [CrossRef]
  17. Wang, P.; Zhao, Y.; Li, Z.; Hsu, C.-C.; Liu, X.; Fu, L.; Hou, Y.-J.; Du, Y.; Xie, S.; Zhang, C.; et al. Reciprocal Regulation of the TOR Kinase and ABA Receptor Balances Plant Growth and Stress Response. Mol. Cell 2018, 69, 100–112.e106. [Google Scholar] [CrossRef] [PubMed]
  18. Iyer, L.M.; Koonin, E.V.; Aravind, L. Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily. Proteins 2001, 43, 134–144. [Google Scholar] [CrossRef]
  19. Ma, Y.; Szostkiewicz, I.; Korte, A.; Moes, D.; Yang, Y.; Christmann, A.; Grill, E. Regulators of PP2C phosphatase activity function as abscisic acid sensors. Science 2009, 324, 1064–1068. [Google Scholar] [CrossRef]
  20. Park, S.Y.; Fung, P.; Nishimura, N.; Jensen, D.R.; Fujii, H.; Zhao, Y.; Lumba, S.; Santiago, J.; Rodrigues, A.; Chow, T.F.; et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science 2009, 324, 1068–1071. [Google Scholar] [CrossRef]
  21. Zhao, Y.; Chan, Z.; Gao, J.; Xing, L.; Cao, M.; Yu, C.; Hu, Y.; You, J.; Shi, H.; Zhu, Y.; et al. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proc. Natl. Acad. Sci. USA 2016, 113, 1949–1954. [Google Scholar] [CrossRef]
  22. Santiago, J.; Rodrigues, A.; Saez, A.; Rubio, S.; Antoni, R.; Dupeux, F.; Park, S.Y.; Márquez, J.A.; Cutler, S.R.; Rodriguez, P.L. Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade A PP2Cs. Plant J. Cell Mol. Biol. 2009, 60, 575–588. [Google Scholar] [CrossRef]
  23. Quan, W.; Hu, Y.; Mu, Z.; Shi, H.; Chan, Z. Overexpression of AtPYL5 under the control of guard cell specific promoter improves drought stress tolerance in Arabidopsis. Plant Physiol. Biochem. PPB 2018, 129, 150–157. [Google Scholar] [CrossRef]
  24. Dittrich, M.; Mueller, H.M.; Bauer, H.; Peirats-Llobet, M.; Rodriguez, P.L.; Geilfus, C.M.; Carpentier, S.C.; Al Rasheid, K.A.S.; Kollist, H.; Merilo, E.; et al. The role of Arabidopsis ABA receptors from the PYR/PYL/RCAR family in stomatal acclimation and closure signal integration. Nat. Plants 2019, 5, 1002–1011. [Google Scholar] [CrossRef]
  25. Zhao, Y.; Xing, L.; Wang, X.; Hou, Y.J.; Gao, J.; Wang, P.; Duan, C.G.; Zhu, X.; Zhu, J.K. The ABA receptor PYL8 promotes lateral root growth by enhancing MYB77-dependent transcription of auxin-responsive genes. Sci. Signal. 2014, 7, ra53. [Google Scholar] [CrossRef]
  26. Li, H.; Gao, Z.; Chen, Q.; Li, Q.; Luo, M.; Wang, J.; Hu, L.; Zahid, M.S.; Wang, L.; Zhao, L.; et al. Grapevine ABA receptor VvPYL1 regulates root hair development in Transgenic Arabidopsis. Plant Physiol. Biochem. PPB 2020, 149, 190–200. [Google Scholar] [CrossRef] [PubMed]
  27. Yadav, S.K.; Santosh Kumar, V.V.; Verma, R.K.; Yadav, P.; Saroha, A.; Wankhede, D.P.; Chaudhary, B.; Chinnusamy, V. Genome-wide identification and characterization of ABA receptor PYL gene family in rice. BMC Genom. 2020, 21, 676. [Google Scholar] [CrossRef]
  28. Bai, G.; Yang, D.H.; Zhao, Y.; Ha, S.; Yang, F.; Ma, J.; Gao, X.S.; Wang, Z.M.; Zhu, J.K. Interactions between soybean ABA receptors and type 2C protein phosphatases. Plant. Mol. Biol. 2013, 83, 651–664. [Google Scholar] [CrossRef] [PubMed]
  29. Gordon, C.S.; Rajagopalan, N.; Risseeuw, E.P.; Surpin, M.; Ball, F.J.; Barber, C.J.; Buhrow, L.M.; Clark, S.M.; Page, J.E.; Todd, C.D.; et al. Characterization of Triticum aestivum Abscisic Acid Receptors and a Possible Role for These in Mediating Fusairum Head Blight Susceptibility in Wheat. PLoS ONE 2016, 11, e0164996. [Google Scholar] [CrossRef]
  30. Fan, W.; Zhao, M.; Li, S.; Bai, X.; Li, J.; Meng, H.; Mu, Z. Contrasting transcriptional responses of PYR1/PYL/RCAR ABA receptors to ABA or dehydration stress between maize seedling leaves and roots. BMC Plant Biol. 2016, 16, 99. [Google Scholar] [CrossRef]
  31. Yu, J.; Yang, L.; Liu, X.; Tang, R.; Wang, Y.; Ge, H.; Wu, M.; Zhang, J.; Zhao, F.; Luan, S.; et al. Overexpression of Poplar Pyrabactin Resistance-Like Abscisic Acid Receptors Promotes Abscisic Acid Sensitivity and Drought Resistance in Transgenic Arabidopsis. PLoS ONE 2016, 11, e0168040. [Google Scholar] [CrossRef]
  32. Guo, D.; Zhou, Y.; Li, H.-L.; Zhu, J.-H.; Wang, Y.; Chen, X.-T.; Peng, S.-Q. Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree. Sci. Rep. 2017, 7, 45157. [Google Scholar] [CrossRef] [PubMed]
  33. Chai, Y.-m.; Jia, H.-f.; Li, C.-l.; Dong, Q.-h.; Shen, Y.-y. FaPYR1 is involved in strawberry fruit ripening. J. Exp. Bot. 2011, 62, 5079–5089. [Google Scholar] [CrossRef]
  34. Zhang, G.; Lu, T.; Miao, W.; Sun, L.; Tian, M.; Wang, J.; Hao, F. Genome-wide identification of ABA receptor PYL family and expression analysis of PYLs in response to ABA and osmotic stress in Gossypium. PeerJ 2017, 5, e4126. [Google Scholar] [CrossRef]
  35. Hou, H.; Lv, L.; Huo, H.; Dai, H.; Zhang, Y. Genome-Wide Identification of the ABA Receptors Genes and Their Response to Abiotic Stress in Apple. Plants 2020, 9, 1028. [Google Scholar] [CrossRef]
  36. Liu, X.; Zhao, X.; Yan, Y.; Shen, M.; Feng, R.; Wei, Q.; Zhang, L.; Zhang, M. Genome-wide analysis of the PYL gene family and identification of PYL genes that respond to cold stress in Triticum monococcum L. Subsp. Aegilopoides. Sci. Rep. 2024, 14, 26627. [Google Scholar] [CrossRef]
  37. Sun, Y.; Xiao, W.; Wang, Q.-n.; Wang, J.; Kong, X.-d.; Ma, W.-h.; Liu, S.-x.; Ren, P.; Xu, L.-n.; Zhang, Y.-J. Multiple variation patterns of terpene synthases in 26 maize genomes. BMC Genom. 2023, 24, 46. [Google Scholar] [CrossRef]
  38. Hufford, M.B.; Seetharam, A.S.; Woodhouse, M.R.; Chougule, K.M.; Ou, S.; Liu, J.; Ricci, W.A.; Guo, T.; Olson, A.; Qiu, Y.; et al. De novo assembly, annotation, and comparative analysis of 26 diverse maize genomes. Science 2021, 373, 655–662. [Google Scholar] [CrossRef]
  39. Shi, T.; Zhang, X.; Hou, Y.; Jia, C.; Dan, X.; Zhang, Y.; Jiang, Y.; Lai, Q.; Feng, J.; Feng, J.; et al. The super-pangenome of Populus unveils genomic facets for its adaptation and diversification in widespread forest trees. Mol. Plant 2024, 17, 725–746. [Google Scholar] [CrossRef] [PubMed]
  40. Jiao, P.; Wu, Z.; Wang, X.; Jiang, Z.; Wang, Y.; Liu, H.; Qin, R.; Li, Z. Short-term transcriptomic responses of Populus euphratica roots and leaves to drought stress. J. For. Res. 2021, 32, 841–853. [Google Scholar] [CrossRef]
  41. Ottow, E.A.; Polle, A.; Brosché, M.; Kangasjärvi, J.; Dibrov, P.; Zörb, C.; Teichmann, T. Molecular characterization of PeNhaD1: The first member of the NhaD Na+/H+ antiporter family of plant origin. Plant Mol. Biol. 2005, 58, 75–88. [Google Scholar] [CrossRef]
  42. Ma, T.; Wang, J.; Zhou, G.; Yue, Z.; Hu, Q.; Chen, Y.; Liu, B.; Qiu, Q.; Wang, Z.; Zhang, J.; et al. Genomic insights into salt adaptation in a desert poplar. Nat. Commun. 2013, 4, 2797. [Google Scholar] [CrossRef]
  43. Sun, J.; Xu, J.; Qiu, C.; Zhai, J.; Zhang, S.; Zhang, X.; Wu, Z.; Li, Z. The chromosome-scale genome and population genomics reveal the adaptative evolution of Populus pruinosa to desertification environment. Hortic. Res. 2024, 11, uhae034. [Google Scholar] [CrossRef] [PubMed]
  44. Sang, Y.; Long, Z.; Dan, X.; Feng, J.; Shi, T.; Jia, C.; Zhang, X.; Lai, Q.; Yang, G.; Zhang, H.; et al. Genomic insights into local adaptation and future climate-induced vulnerability of a keystone forest tree in East Asia. Nat. Commun. 2022, 13, 6541. [Google Scholar] [CrossRef] [PubMed]
  45. Long, Z.; Sang, Y.; Feng, J.; Zhang, X.; Shi, T.; Zhang, L.; Mao, K.; Rieseberg, L.H.; Liu, J.; Wang, J. Evolutionary genomics predicts adaptive genetic and plastic gene expression responses to climate change in a key alpine forest tree species. bioRxiv 2024. [Google Scholar] [CrossRef]
  46. Chen, L.; Jiang, Y.; Shi, T.; Jia, C.; Long, Z.; Zhang, X.; Sang, Y.; Liu, J.; Wang, J. Chromosomal-level genome assembly of Populus davidiana. bioRxiv 2023. [Google Scholar] [CrossRef]
  47. Schiffthaler, B.; Delhomme, N.; Bernhardsson, C.; Jenkins, J.; Jansson, S.; Ingvarsson, P.; Schmutz, J.; Street, N. An Improved Genome Assembly of the European Aspen Populus tremula. bioRxiv 2019. [Google Scholar] [CrossRef]
  48. Zhang, L.; Zhao, J.; Bi, H.; Yang, X.; Zhang, Z.; Su, Y.; Li, Z.; Zhang, L.; Sanderson, B.J.; Liu, J.; et al. Bioinformatic analysis of chromatin organization and biased expression of duplicated genes between two poplars with a common whole-genome duplication. Hortic. Res. 2021, 8, 62. [Google Scholar] [CrossRef]
  49. Li, Y.; Wang, D.; Wang, W.; Yang, W.; Gao, J.; Zhang, W.; Shan, L.; Kang, M.; Chen, Y.; Ma, T. A chromosome-level Populus qiongdaoensis genome assembly provides insights into tropical adaptation and a cryptic turnover of sex determination. Mol. Ecol. 2023, 32, 1366–1380. [Google Scholar] [CrossRef]
  50. Liu, S.; Wang, Z.; Shi, T.; Dan, X.; Zhang, Y.; Liu, J.; Wang, J. Chromosomal-level genome assembly of Populus adenopoda. bioRxiv 2023. [Google Scholar] [CrossRef]
  51. Li, J.; Shi, C.; Sun, D.; He, Y.; Lai, C.; Lv, P.; Xiong, Y.; Zhang, L.; Wu, F.; Tian, C. The HAB1 PP2C is inhibited by ABA-dependent PYL10 interaction. Sci. Rep. 2015, 5, 10890. [Google Scholar] [CrossRef]
  52. Hao, Q.; Yin, P.; Li, W.; Wang, L.; Yan, C.; Lin, Z.; Wu, J.Z.; Wang, J.; Yan, S.F.; Yan, N. The Molecular Basis of ABA-Independent Inhibition of PP2Cs by a Subclass of PYL Proteins. Mol. Cell 2011, 42, 662–672. [Google Scholar] [CrossRef]
  53. Sun, L.; Wang, Y.-P.; Chen, P.; Ren, J.; Ji, K.; Li, Q.; Li, P.; Dai, S.-J.; Leng, P. Transcriptional regulation of SlPYL, SlPP2C, and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress. J. Exp. Bot. 2011, 62, 5659–5669. [Google Scholar] [CrossRef] [PubMed]
  54. Zhan, X.; Lu, Y.; Zhu, J.K.; Botella, J.R. Genome editing for plant research and crop improvement. J. Integr. Plant Biol. 2021, 63, 3–33. [Google Scholar] [CrossRef] [PubMed]
  55. Cannon, S.B.; Mitra, A.; Baumgarten, A.; Young, N.D.; May, G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004, 4, 10. [Google Scholar] [CrossRef]
  56. Tuskan, G.A.; Difazio, S.; Jansson, S.; Bohlmann, J.; Grigoriev, I.; Hellsten, U.; Putnam, N.; Ralph, S.; Rombauts, S.; Salamov, A.; et al. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 2006, 313, 1596–1604. [Google Scholar] [CrossRef]
  57. Tian, X.; Wang, Z.; Li, X.; Lv, T.; Liu, H.; Wang, L.; Niu, H.; Bu, Q. Characterization and Functional Analysis of Pyrabactin Resistance-Like Abscisic Acid Receptor Family in Rice. Rice 2015, 8, 28. [Google Scholar] [CrossRef]
  58. Zhang, Y.; Yang, Z.; He, Y.; Liu, D.; Liu, Y.; Liang, C.; Xie, M.; Jia, Y.; Ke, Q.; Zhou, Y.; et al. Structural variation reshapes population gene expression and trait variation in 2,105 Brassica napus accessions. Nat. Genet. 2024, 56, 2538–2550. [Google Scholar] [CrossRef]
  59. Zhao, K.; Xue, H.; Li, G.; Chitikineni, A.; Fan, Y.; Cao, Z.; Dong, X.; Lu, H.; Zhao, K.; Zhang, L.; et al. Pangenome analysis reveals structural variation associated with seed size and weight traits in peanut. Nat. Genet. 2025, 57, 1250–1261. [Google Scholar] [CrossRef]
  60. Li, Z.; Fu, D.; Wang, X.; Zeng, R.; Zhang, X.; Tian, J.; Zhang, S.; Yang, X.; Tian, F.; Lai, J.; et al. The transcription factor bZIP68 negatively regulates cold tolerance in maize. Plant Cell 2022, 34, 2833–2851. [Google Scholar] [CrossRef] [PubMed]
  61. Fuentes, R.R.; Chebotarov, D.; Duitama, J.; Smith, S.; De la Hoz, J.F.; Mohiyuddin, M.; Wing, R.A.; McNally, K.L.; Tatarinova, T.; Grigoriev, A.; et al. Structural variants in 3000 rice genomes. Genome Res. 2019, 29, 870–880. [Google Scholar] [CrossRef] [PubMed]
  62. Zhao, H.; Wang, X.; Jia, Y.; Minkenberg, B.; Wheatley, M.; Fan, J.; Jia, M.H.; Famoso, A.; Edwards, J.D.; Wamishe, Y.; et al. The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance. Nat. Commun. 2018, 9, 2039. [Google Scholar] [CrossRef]
  63. Hao: Structural Biological Studies of the Abscisic Acid Receptor PYL Protein Family. Tsinghua University. Issue 9. 2015. Available online: https://kns.cnki.net/ (accessed on 8 April 2025).
  64. Fernández-Cancelo, P.; Muñoz, P.; Echeverría, G.; Larrigaudière, C.; Teixidó, N.; Munné-Bosch, S.; Giné-Bordonaba, J. Ethylene and abscisic acid play a key role in modulating apple ripening after harvest and after cold-storage. Postharvest Biol. Technol. 2022, 188, 111902. [Google Scholar] [CrossRef]
  65. Zhao, H.; Nie, K.; Zhou, H.; Yan, X.; Zhan, Q.; Zheng, Y.; Song, C.-P. ABI5 modulates seed germination via feedback regulation of the expression of the PYR/PYL/RCAR ABA receptor genes. New Phytologist. 2020, 228, 596–608. [Google Scholar] [CrossRef]
  66. Ramya, M.; Kwon, O.K.; An, H.R.; Park, P.M.; Baek, Y.S.; Park, P.H. Floral scent: Regulation and role of MYB transcription factors. Phytochem. Lett. 2017, 19, 114–120. [Google Scholar] [CrossRef]
  67. Lau, S.E.; Schwarzacher, T.; Othman, R.Y.; Harikrishna, J.A. dsRNA silencing of an R2R3-MYB transcription factor affects flower cell shape in a Dendrobium hybrid. BMC Plant Biol. 2015, 15, 194. [Google Scholar] [CrossRef]
  68. Wang, H.X.; Liu, T.Y.; Zhuang, W.B.; Wang, Z.Z.L.; Qu, S.C.; Zhai, H.H. Research advances in the function of anthocyanin in plant stress response. J. Agric. Biotechnol. 2020, 28, 174–183. [Google Scholar]
  69. Di, F.; Jian, H.; Wang, T.; Chen, X.; Ding, Y.; Du, H.; Lu, K.; Li, J.; Liu, L. Genome-Wide Analysis of the PYL Gene Family and Identification of PYL Genes That Respond to Abiotic Stress in Brassica napus. Genes 2018, 9, 156. [Google Scholar] [CrossRef]
  70. Li, Y.; Wang, D.; Sun, C.; Hu, X.; Mu, X.; Hu, J.; Yang, Y.; Zhang, Y.; Xie, C.G.; Zhou, X. Molecular characterization of an AtPYL1-like protein, BrPYL1, as a putative ABA receptor in Brassica rapa. Biochem. Biophys. Res. Commun. 2017, 487, 684–689. [Google Scholar] [CrossRef] [PubMed]
  71. Asad, M.A.U.; Zakari, S.A.; Zhao, Q.; Zhou, L.; Ye, Y.; Cheng, F. Abiotic Stresses Intervene with ABA Signaling to Induce Destructive Metabolic Pathways Leading to Death: Premature Leaf Senescence in Plants. Int. J. Mol. Sci. 2019, 20, 256. [Google Scholar] [CrossRef] [PubMed]
  72. Mistry, J.; Finn, R.D.; Eddy, S.R.; Bateman, A.; Punta, M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 2013, 41, e121. [Google Scholar] [CrossRef]
  73. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef] [PubMed]
  74. Wang, D.; Zhang, Y.; Zhang, Z.; Zhu, J.; Yu, J. KaKs_Calculator 2.0: A toolkit incorporating gamma-series methods and sliding window strategies. Genom. Proteom. Bioinform. 2010, 8, 77–80. [Google Scholar] [CrossRef] [PubMed]
  75. Chen, C.; Xia, R.; Chen, H.; He, Y. TBtools, a Toolkit for Biologists integrating various HTS-data handling tools with a user-friendly interface. bioRxiv 2018, 289660. [Google Scholar] [CrossRef]
  76. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef]
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Figure 1. Phylogenetic analysis of PYL genes in Populus. An unrooted Neighbor-Joining phylogenetic tree was constructed from Arabidopsis and 30 PopPYL. The bootstrap test was set to 1000 replicates. Different colors represent different subfamilies of the PYL gene.
Figure 1. Phylogenetic analysis of PYL genes in Populus. An unrooted Neighbor-Joining phylogenetic tree was constructed from Arabidopsis and 30 PopPYL. The bootstrap test was set to 1000 replicates. Different colors represent different subfamilies of the PYL gene.
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Plants 14 02541 g002
Figure 2. Distribution of the PYL genes in the reference genome (A) and non-reference genome (B) genome. PYL21-PYL30 were species-specific genes. The left axis shows the length of each chromosome and contig, as was estimated in megabases (Mb).
Figure 2. Distribution of the PYL genes in the reference genome (A) and non-reference genome (B) genome. PYL21-PYL30 were species-specific genes. The left axis shows the length of each chromosome and contig, as was estimated in megabases (Mb).
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Plants 14 02541 g003
Figure 3. Comparison of the conserved motifs and gene structures of intermediate clade genes PopPYL6 (A), PopPYL7 (B), and PopPYL13 (C) in 17 Populus. Left panel: the phylogenetic tree was constructed with the full length of PYL proteins using MEGA 7.0. Middle panel: the conserved motifs for PYL proteins. Motifs 1–10 are highlighted with different colors. Right panel: exon–intron structure of PYL genes.
Figure 3. Comparison of the conserved motifs and gene structures of intermediate clade genes PopPYL6 (A), PopPYL7 (B), and PopPYL13 (C) in 17 Populus. Left panel: the phylogenetic tree was constructed with the full length of PYL proteins using MEGA 7.0. Middle panel: the conserved motifs for PYL proteins. Motifs 1–10 are highlighted with different colors. Right panel: exon–intron structure of PYL genes.
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Plants 14 02541 g004
Figure 4. Ka/Ks values of PopPYL. (A) Synteny analysis of PYL among P. euphratica and other plant species. (B) Distribution of Ka/Ks values of PopPYL in 17 Populus varieties. (C) Heatmap of the frequency of occurrence of different Populus varieties at each PYL with a Ka/Ks ratio > 1.
Figure 4. Ka/Ks values of PopPYL. (A) Synteny analysis of PYL among P. euphratica and other plant species. (B) Distribution of Ka/Ks values of PopPYL in 17 Populus varieties. (C) Heatmap of the frequency of occurrence of different Populus varieties at each PYL with a Ka/Ks ratio > 1.
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Plants 14 02541 g005
Figure 5. (A) Predicted cis-elements in promoter regions of PYL_EUP and PYL_PRU genes. (B) Cis-regulatory elements in PYL promoters of EUP and PRU. The depth of red square indicates the number of cis-acting elements for each gene, while white square indicates 0.
Figure 5. (A) Predicted cis-elements in promoter regions of PYL_EUP and PYL_PRU genes. (B) Cis-regulatory elements in PYL promoters of EUP and PRU. The depth of red square indicates the number of cis-acting elements for each gene, while white square indicates 0.
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Plants 14 02541 g006
Figure 6. Effect of SVs on genes. (A) The effects of SV insertion and deletion on PYL_EUP. (B) The expression of PYL_EUP was significantly affected by SVs. (* p < 0.05).
Figure 6. Effect of SVs on genes. (A) The effects of SV insertion and deletion on PYL_EUP. (B) The expression of PYL_EUP was significantly affected by SVs. (* p < 0.05).
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Plants 14 02541 g007
Figure 7. The expression pattern of PYL genes under drought stress was analyzed by heat mapping. (A) Pe-leaf vs. Pe-stem vs. Pe-root; (B) PeDT-leaf vs. PeCK-leaf; (C) PeDT-stem vs. PeCK-stem; (D) PeDT-root vs. PeCK-root.
Figure 7. The expression pattern of PYL genes under drought stress was analyzed by heat mapping. (A) Pe-leaf vs. Pe-stem vs. Pe-root; (B) PeDT-leaf vs. PeCK-leaf; (C) PeDT-stem vs. PeCK-stem; (D) PeDT-root vs. PeCK-root.
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Plants 14 02541 g008
Figure 8. Expression of PYL genes analyzed by RT-qPCR in different tissues of P. euphratica. The bars are standard deviations (SDs) of three biological replicates.
Figure 8. Expression of PYL genes analyzed by RT-qPCR in different tissues of P. euphratica. The bars are standard deviations (SDs) of three biological replicates.
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Table 1. Statistics of data sources.
Table 1. Statistics of data sources.
SpeciesAbbreviationAssembly Size (Mb)Genome DataReference
Populus euphraticaEUP518.6Unpublished
Populus pruinosaPRU521.1https://www.ncbi.nlm.nih.gov/search/all/?term=PRJNA863418Pprgenome_fa/20705107/2[43]
Populus pseudoglaucaPSE448.7Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUF00000000		[39]
Populus wuanaWUA417.4Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUE00000000		[39]
Populus szechuanicaSZE429.1Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUK00000000		[39]
Populus yunnanensisYUN433.7Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUD00000000		[39]
Populus koreanaKOR401.4Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBHRS00000000		[44]
Populus trichocarpa v4.1TRI392.2Database: https://phytozome-next.jgi.doe.gov/Phytozome
Populus deltoids v2.1DEL446.8Database: https://phytozome-next.jgi.doe.gov/Phytozome
Populus simoniiSIM408.0Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJTX00000000		[39]
Populus lasiocarpaLAS419.5Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUB00000000		[45]
Populus davidianaDAV441.1Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJTW00000000		[46]
Populus rotundifoliaROT414.3Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJUA00000000		[39]
Populus tremulaTRE408.8Database: http://popgenie.org[47]
Populus alba var. pyramidalisALB408.1Database: http://bigd.big.ac.cn/bioproject;
Accession number: PRJCA002423		[48]
Populus qiongdaoensisQIO391.3Database: http://bigd.big.ac.cn/bioproject;
Accession number: PRJCA007862		[49]
Populus adenopodaADE383.4Database: https://bigd.big.ac.cn/gwh;
Accession number: GWHBJTV00000000		[50]
Note: URL (Accessed on: 8 April 2025).
Table 2. Summary of PYL protein properties.
Table 2. Summary of PYL protein properties.
Sequence IDNumber of Amino AcidMolecular WeightTheoretical pIInstability IndexAliphatic Index
IPYL154~1899673.47~21,213.945.89~10.1738.12~43.181.9~110
PYL2190~20521,377.46~2,3149.565.6~7.7334.04~39.9688.53~91.63
PYL370677,272.845.2230.4885.44
PYL4169~24818,686~27,574.394.44~5.1526.15~44.6266.73~77.65
PYL2420222,653.534.747.6574.75
PYL14168~38118,895.38~43,039.844.46~4.825.8~31.7372.66~78.99
PYL2118821,413.68.4434.0381.38
PYL19108~14312,205.97~16,086.064.39~4.5940.25~43.8876.36~92.96
PYL15169~32918,665.8~36,424.994.71~5.5529.9~42.9172.12~86.8
PYL18102~11511,253.82~12,760.65.57~5.8626.7~36.8577.91~85
PYL12103~16311,430.91~18,163.624.46~4.923.22~45.1977.04~93.74
PYL2916318,053.434.6429.9486.01
PYL2618620,986.816.4439.1584.25
PYL861567,740.227.2338.3893.07
PYL3019021,527.636.0638.5591.63
PYL2519021,487.526.0640.0189.05
PYL2719021,463.466.0640.3188.53
PYL2322024,606.145.6243.1792
PYL13191~22921,355.14~25,681.55.78~6.1140.64~48.9694.29~98.25
PYL2819121,369.165.7848.9694.29
PYL16134~28215,264.5~32,181.385.55~8.0338.94~44.9195.9~100.9
IIPYL6186~22320,570.31~24,181.238.5~9.2142.26~46.9180.59~85.02
PYL17214~22223,268.13~24,100.997.66~8.2650.16~54.1479.41~80.56
PYL2230634,088.896.346.7893.24
PYL10214~23823,103.88~26,319.767.11~9.2238.54~51.1378.35~85.47
PYL1121423,086.16~23,310.387.1~7.6849.26~54.6986.82~90.93
IIIPYL5124~19113,517.37~21,341.065.2~7.012.05~51.3982.43~89.69
PYL9203~21022,319.97~23,1904.93~5.5332.74~42.882.1~86.85
PYL7201~25922,392.17~28,658.255.08~5.6229.23~34.5879.57~84.33
PYL2016918,961.56~18,961.585.9438.88~40.0285.27~85.86
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Han, X.; Qiu, C.; Gai, Z.; Zhai, J.; Song, J.; Sun, J.; Li, Z. Pan-Genome-Based Characterization of the PYL Transcription Factor Family in Populus. Plants 2025, 14, 2541. https://doi.org/10.3390/plants14162541

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Han X, Qiu C, Gai Z, Zhai J, Song J, Sun J, Li Z. Pan-Genome-Based Characterization of the PYL Transcription Factor Family in Populus. Plants. 2025; 14(16):2541. https://doi.org/10.3390/plants14162541

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Han, Xiaoli, Chen Qiu, Zhongshuai Gai, Juntuan Zhai, Jia Song, Jianhao Sun, and Zhijun Li. 2025. "Pan-Genome-Based Characterization of the PYL Transcription Factor Family in Populus" Plants 14, no. 16: 2541. https://doi.org/10.3390/plants14162541

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Han, X., Qiu, C., Gai, Z., Zhai, J., Song, J., Sun, J., & Li, Z. (2025). Pan-Genome-Based Characterization of the PYL Transcription Factor Family in Populus. Plants, 14(16), 2541. https://doi.org/10.3390/plants14162541

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