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Genome-wide identification of pyrabactin resistance 1-like (PYL) gene family under phytohormones and drought stresses in alfalfa (Medicago sativa)

BMC Genomics volume 26, Article number: 383 (2025) Cite this article

Abstract

Background

The Pyrabactin resistance 1-like proteins (PYR/PYL/RCAR) protein plays a critical regulatory role in the ABA signal transduction pathway as a direct receptor of abscisic acid (ABA). Although PYL genes have been identified in a variety of plants, the evolution and structural characteristics of these genes in alfalfa (Medicago sativa) are largely unknown. Therefore, a comprehensive bioinformatics analysis of the PYL gene family was performed in this research.

Results

The results indicated that 41 MsPYL genes were unevenly distributed across 24 chromosomes. According to gene structure, conservative features, and phylogenetic relationships, MsPYL proteins can be divided into 6 groups, all of which have PYR/PYL/RCAR domains, and MsPYL proteins are relatively small (molecular weight 19.59 kDa to 25.31 kDa). MsPYL genes contains cis-acting elements that has functions in plant growth and development, hormone regulation, and stress response. Furthermore, transcriptome data showed that drought stress affected the MsPYL genes’ expression levels in alfalfa. Tissue specificity analysis revealed that all MsPYL genes exhibited varying responses to drought, abscisic acid (ABA), salicylic acid (SA), and indole-3-acetic acid (IAA). Additionally, all MsPYL genes were expressed to different extents in both the aboveground and underground tissues following stimulation. They were induced by IAA, ABA, and SA from 6 h to 12 h, and ABA induced MsPYL4 most significantly at the 12 h mark. MsPYL4, MsPYL8, MsPYL11, and MsPYL19 were expressed only after hormone treatment.

Conclusions

The results of this study indicate that the MsPYL genes are closely related to stress response and provide new candidate genes for further exploration of MsPYL genes function and improvement and innovation of drought-resistant alfalfa germplasm.

Peer Review reports

Background

Protein phosphatase 2 C (PP2C) and protein phosphatase 1-related protein kinase 2 (SnRK2) are two key components of the ABA signaling pathway mediated by PYR/PYL/RCAR [1]. Together, they form a dual negative regulatory system (PYR/PYL/RCAR (+ ABA) ┤PP2C ┤SnRK2) that regulate the expression of downstream genes in the ABA signaling pathway (Fig. 1). This process occurs in the cytoplasm, nucleus, and plasma membrane. The PYR/PYL/RCAR receptor protein exists as an inactive dimer when the abscisic acid (ABA)level is low; PP2C proteins dephosphorylate SnRK2 in this state, leading to the inactivation of SnRK2 and the formation of a complex [2–3]. When the concentration of ABA in plants increases rapidly in response to drought conditions, it subsequently induces structural changes in the ABA receptor (Pyrabactin resilience-like 1, PYL) [4]. They can bind to PP2Cs after structural changes in ABA receptors, thereby diminishing the inhibitory effect of PP2Cs on SnRK2. Subsequently, the activated SnRK2 can subsequently regulate the expression of downstream stress-responsive genes, thereby enhancing the plant’s stress tolerance [5–6]. In addition, activated SnRK2 acts on related membrane proteins, such as the activation of anion channels. This activation can facilitate the release of intracellular anions, leading to an increase in pH and subsequently activating outward potassium channels, ultimately inducing stomatal closure [5, 7].

Many plants have been researched for the PYL gene family, and the model plant Arabidopsis (Arabidopsis thaliana) has been used to validate its gene functions [8–9]. It was discovered that the overexpression of AtPYL4 was highly sensitive to ABA and can enhance water use efficiency [10–11]. Similarly, AtPYL9 has improved drought resistance and delayed leaf senescence in both A. thaliana and O.sativa [8]. Overexpression of OsPYL3, OsPYL11, and OsPYL5 increased sensitivity to changes in ABA content while simultaneously improving tolerance to water deficit in rice [12–13]. TaPYL4 overexpression not only has increased the sensitivity of plants to ABA but also reduced water consumption in wheat, thereby preserving yield in the event of a water shortage [9]. Furthermore, GhPYL also promotes root growth in cotton (Gossypium hirsutum) seedlings [14]. Drought stress can lead to an increase in ABA content in plants, which is detected by the ABA receptor PYR1/PYL/RCAR. At the same time, ABA signaling mediates actin remodeling to regulate stomatal opening and closing during drought conditions [15]. Additionally, the CKL2 kinase can also influence ABA signal transduction during this phase by giving PYL, ABI1, and ABI2 feedback control [16]. The osmotic tolerance, ABA sensitivity, and drought tolerance of the transgenic plants were significantly increased by the heterologous overexpression of foreign genes, such as cotton GhPYL and Brachypodium distachyon BdPYL in A. thaliana [14, 17].

A. thaliana AtPYL plays a crucial role in regulating ABA-mediated seed germination and seedling development [18], the process influenced by the bZIP transcription factor AtABI5 [19]. ABA receptors in A. thaliana positively contribute to resistance against extreme temperatures [12]. Similarly, Liriodendron chinense (LchiPYL) exhibits a favorable response to cold stress, ABA, and light treatment [20]. In the research on cassava (Manihot esculenta), it was found that the level of MePYL was induced by both salt stress and oxidative conditions [21]. The expression of the NtPYL4 in tobacco (Nicotiana tabacum) is regulated by jasmonic acid (JA), indicating that NtPYL4 may mediate the interplay between JA signaling and ABA signaling [22]. The above research results indicated that the PYL genes play a critical role in regulating plant growth and enhancing resistance [23]. However, little is known about the alfalfa ABA receptor genes PYL, which significantly limits our understanding of the ABA signal transduction mechanisms and the regulatory processes involved in stress response in alfalfa.

Alfalfa, a high-quality leguminous forage, possesses a well-developed root system and exhibits strong drought resistance compared to others [24], however, drought remains a limiting factor affecting alfalfa cultivation and production. Up to now, numerous studies on the transcriptome sequencing of alfalfa have been reported [25,26,27]. These studies primarily encompass abiotic stress, biotic stress, various tissues, different traits, and full-length transcriptome sequencing. In recent years, research on drought resistance genes in alfalfa has mainly focused on the regulation of transcription factors and gene expression. By cloning and expressing resistance-related genes, the molecular mechanisms underlying alfalfa’s resistance have been further elucidated, with the potential for application in molecular improvement. This study used bioinformatics methods to identify the PYL gene family in alfalfa and analyzed its systematic evolution, gene structure, chromosome localization, collinearity, and family cis-acting elements and expression. This offers a theoretical framework for investigating the role of PYL genes in alfalfa further and developing novel, stress-resistant cultivars.

Materials and methods

Identification and chromosomal distribution of PYL gene family members in alfalfa

PYL proteins sequence files (Fasta and GFF) of 14 A. thaliana, 42 O.sativa, and 48 M.truncatula (https://plants.ensembl.org/index.html) were obtained from the NCBI website. Subsequently, a blast alignment (E-value < 1e-5) of the PYL proteins from these three species was performed. To verify the accuracy of the obtained PYL proteins sequences, the online tool Pfam Program (https://pfam.xfam.org) was used to check for the presence of any domains within these sequences (E-value < 1e-5). After deleting the protein sequences that lack the PYL domain, redundant sequences in the remaining PYL proteins sequences were removed using the CD-Hit (Cluster Database at High Identity with Tolerance) tool (http://www.bioinformatics.org/cd-hit/). Finally, 41 MsPYL genes were identified in alfalfa. These genes were visualized on chromosomes using TBtools (v2.142) and were named MsPYL1 to MsPYL41 based on their chromosomal positions [28]. Subsequently, the physicochemical properties of the MsPYL proteins were analyzed by the tool ProParam (http://web.expasy.org/proparam). To predict the potential functions of the MsPYL genes, the Blast-II (v 2.096) software was employed to annotate the functions of the MsPYL protein sequences [29].

Analysis of the protein secondary structural of the MsPYL protein

The online analysis tool SOPMA (https://npsa.lyon.inserm.fr/) was utilized to predict the protein secondary structure of the MsPYL protein.

Analysis of the physicochemical properties of the MsPYL gene

The physicochemical properties of the identified MsPYLs cDNA sequences were obtained from the GFF file of the reference genome of alfalfa using the TBtools (v2.142). The subcellular localization of the MsPYLs proteins was predicted through the online tool WoLF PSORT (https://wolfpsort.hgc.jp/), with the highest score being considered as the prediction result.

Conserved motif and evolutionary analysis of the MsPYL gene

Using the online tool MEME (Multiple Experience Maximization for Motif Elicitation), the conserved motif of the MsPYL genes were identified (http://meme-suite.org/index.html). Utilizing Clustal W (http://www.clustal.org/clustal2), compare the protein sequences of these genes to predict their evolutionary relationships. The phylogenetic tree of MsPYL, MtPYL, and AtPYL was constructed using the Neighbor-Joining (NJ) method [30]. The specific parameters included the Poisson model, with the bootstrap value set to 1000, while the remaining parameters were left at their defaults. The MEME online program (https://meme-suite.org/meme/index.html) was used to predict the conserved motif in the MsPYL proteins, with the parameters set to their default values and a maximum motif number of 10.0.

MsPYL gene collinearity analysis

MCScanX software (https://github.com/wyp1125/MCScanX) was used to analyze the chromosome collinearity of the MsPYL gene of alfalfa [31–32], and the results were visualized by TBtools (v2.142).

Homeopathic element analysis of MsPYL gene promoter

The upstream 2000 bp sequence of the MsPYL initiation codon was extracted using TBtools, and the cis-acting elements were predicted using the online resource PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Expression pattern analysis of MsPYL

Analyzing the expression patterns of the MsPYL genes family during drought by mining transcriptome data related to drought stress and uploading the relevant transcriptome data to the NCBI (https://www.ncbi.nlm.nih.gov/) website (BioProject ID: PRJNA 1098431).

Tissue-specific expression of the MsPYL gene

Medicago. sativa ‘Qingshui’(QS), M. sativa ‘Longdong’(LD), and M. varia Martin ‘Gongnong’ No. 4 (GN) alfalfa were chosen as the experimental materials. Seeds of uniform size and full integrity were selected, and high-temperature sterilized river sand was used as the culture substrate. They were placed in the growth chamber at 25℃, growth conditions:16 / 8 h light / dark cycle, relative humidity 60%, and 450 mol·m− 2·s− 1 of photosynthetic light flux density from June 5, 2024, to July 10, 2024. A total of 100 mL of Hoagland nutrient solution was applied every 2 days. The seedlings were treated with ABA (100 µmol·L− 1), IAA (100 µmol·L− 1), SA (500 µmol·L− 1), MeJA (500 µmol·L− 1), and PEG-6000 (20%) after 5 weeks of culture, respectively [33]. Root and leaf tissue samples were collected after processing for 0, 6, 12, and 24 hours, respectively. The collected samples were then frozen in liquid nitrogen and stored at -80℃ in a refrigerator.

RNA was extracted using the Plant RNA Kit (Accurate Biotechnology, AG21019-Steady Pure, China), and complementary DNA (cDNA) was synthesized using the Evo M-MLV RT Kit. Primers were designed using Primer 5.0 and subsequently synthesized and purified by Sangon Biotech. Actin 2 was chosen as the internal reference gene (Table 1). RT-qPCR analysis was performed on a Roche LightCycler 96 (Roche LightCycler 96, Roche, Switzerland), following the instructions provided with the 2×Ultra SYBR Green qPCR Mix (CISTRO) kit.

Table 1 Primers used for qRT-PCR
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Statistical analysis

Excel 2019 was utilized to calculate the gene expression data, while SPSS 19.0 software was employed for statistical analysis. Additionally, Origin (Pro 2021) and TBtools (v2.142) were used for graphical representation.

Results

Identification and chromosome distribution of the MsPYL gene

This research utilized a Hidden Markov Model (HMM) and domain analysis to identify and screen the members of the PYL gene family present in the alfalfa genome, resulting in the identification of 41 MsPYL genes. These 41 MsPYL genes were unevenly distributed across 24 of the 32 chromosomes in alfalfa, with no MsPYL genes distribution on chromosomes 2.1 to 2.4 and 6.1 to 6.4. Chromosomes 3.2 and 5.3 contained the highest number of MsPYL genes, with 6 and 5, respectively. In contrast, only one MsPYL gene was found on chromosomes 4.1 to 5.1 and 5.4 to 8.4. Based on the positional order of these genes on the chromosomes, the MsPYL genes were named MsPYL1 to MsPYL41 (Fig. 2). All 41 MsPYL genes were located on various chromosomes according to their chromosomal positions (Supplement Table 1).

Physiochemical characteristics of the MsPYL protein

The PYL family members of alfalfa were further analyzed. The characteristics of the MsPYL proteins, including amino acid count, molecular weight, isoelectric point, and fat solubility index, exhibit significant variation (Table 2). The protein sequence lengths range from 175 (MsPYL6) to 231 amino acids (MsPYL9, MsPYL11, MsPYL17, and MsPYL18). The molecular weights vary from 19.59 (MsPYL6) to 25.31 kDa (MsPYL9, MsPYL11, and MsPYL18). The isoelectric points range from 5.66 (MsPYL6) to 8.50 (MsPYL24, MsPYL26, and MsPYL31). The coefficients of instability range from 32.11 (MsPYL32) to 52.59 (MsPYL12 through MsPYL16), while the lipolytic index ranges from 77.35 (MsPYL32) to 112.40 (MsPYL6). The hydrophilic index was less than 0 for all proteins except MsPYL6, with values ranging from − 0.547 (MsPYL32) to -0.084 (MsPYL24 and MsPYL26). Only 24.39% of the MsPYL proteins were localized in the nucleus, 26.83% in the chloroplast, 39.02% in the cytoplasm, 3 (MsPYL3, MsPYL5, and MsPYL8) in the cytoskeleton, and 1 (MsPYL1) in the cytoskeleton.

Table 2 Basic information analysis of MsPYL gene family
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Phylogenetic evolution and motif analysis of the MsPYL gene

To investigate the evolutionary relationships and classification of the alfalfa PYL family genes, a multiple sequence alignment was conducted on 41 PYL proteins. Fourteen A. thaliana PYL proteins were selected as representative sequences, and the 41 alfalfa PYL protein sequences were categorized into 6 groups (Fig. 3). Group VIB contains the largest number of members (with 28), while groups I, II, III, IV and V comprise 6, 11, 4, 21, and 10, respectively. Phylogenetic analysis revealed that alfalfa and M.truncatula exhibit high homology within the same species, suggesting that some genes may share similar physiological functions.

The conserved motifs of the PYL gene proteins were identified using the website (https://meme-suite.org/meme/index.html), and their sequences and annotations were further predicted using the SMART database. It was found that the MsPYL proteins contained 6 to 9 motifs (Fig. 4). The lengths of the conserved motifs range from approximately 6 to 50 amino acids, with most MsPYL proteins containing 3 conserved motifs (Fig. 4), each ranging from about 41 to 50 amino acids in length. Conserved motifs 1, 2, and 3 are highly conserved across MsPYL1 to MsPYL41, while conserved motif 4 is highly conserved in MsPYL9, MsPYL11, MsPYL17, and MsPYL19 to MsPYL33 (Fig. 4 and Supplement Table 2).

Homeomorphic elements, gene replication, and chromosome collinearity analysis of the MsPYL gene

By analyzing the promoters of 41 upstream sequences of the MsPYL genes (Fig. 5), it was found that they primarily contain response elements related to hormones, light, low temperature, stress, and growth and development. Among them, hormones and light response elements constitute the largest proportion of the MsPYL genes. IAA-responsive elements were identified in MsPYL24, MsPYL26, MsPYL31, and MsPYL33, while MeJA-responsive elements were detected in MsPYL25, MsPYL27 to MsPYL29, MsPYL30, and MsPYL32 in this research. SA response elements constitute the smallest proportion among hormone response elements, having been identified exclusively in MsPYL38. At the same time, the promoter of the MsPYL genes contains ABA, GA, and SA response elements. In conclusion, it is speculated that PYL family genes are closely related to MeJA and may interact with ABA, GA, and SA receptors at the expression level. This suggests that PYL family genes may play a great role in the growth, development, and stress response of alfalfa. Gene replication is crucial for the generation of new genes and functions, and segmental and tandem repeats are important drivers of gene family expansion. It was found that there were 29 tandem repeat events among the 41 members of the MsPYL genes family (Fig. 6). These showed that the evolution of MsPYLs may primarily rely on fragment duplication. Based on this, the collinearity relationship among the PYL genes of M. sativa, A. thaliana, and M. truncatula was also investigated to gain a better understanding of the potential evolutionary processes of the PYL gene family in other crops. It was found that 41 MsPYL genes had collinearity with the genes of A. thaliana and M.truncatula, indicating that the distribution of PYL genes in the three plants was relatively conservative (Fig. 7).

Structure and expression pattern analysis of the MsPYL gene

Functional predictions indicated that the PYR/PYL/RCAR-like domain exists in all PYL genes of alfalfa (Fig. 8). Further analysis of the transcriptome data from previous experiments on alfalfa under drought stress revealed significant upregulation of the PYL gene family. Specifically, the expression levels of MsPYL3, MsPYL20, MsPYL28, and MsPYL29 were markedly increased after QS underwent moderate drought stress. In contrast, MsPYL3, MsPYL20, MsPYL28, and MsPYL29 exhibited only slight expression when LD and GN were subjected to moderate drought stress, respectively (Fig. 9). The expression of MsPYL16 was upregulated in QS under severe stress. MsPYL1 and MsPYL8 were upregulated under moderate and severe stress in LD, while MsPYL27 was upregulated only in GN under severe stress. MsPYL4, MsPYL6, MsPYL15, MsPYL39, and MsPYL40 were all upregulated in response to increasing stress levels in GN, whereas MsPYL26 was exclusively expressed in GN and downregulated as stress intensified. The varying expression levels of the PYL gene in different materials may be attributed to each material’s inherent drought resistance characteristics or to the differing activities of transcription factors that regulate the gene across these materials.

Structural analysis of MsPYL genes

Gene structure analysis indicated that the number of exons in the MsPYL genes ranges from 1 to 4, while the number of introns varies from 0 to 3 (Fig. 10). Notably, MsPYL1, MsPYL3, MsPYL7, MsPYL9, MsPYL11, MsPYL17, MsPYL18, and MsPYL20 through MsPYL37 do not contain any introns. This variation in intron presence may play a crucial role in the evolution of functional diversity within the alfalfa PYL gene family. The online analysis tool SOPMA was utilized to predict the protein secondary structure of the alfalfa PYL gene family (Fig. 11). In the MsPYL proteins, the α-helix content ranges from 39 to 54%, irregular curls range from 31 to 44%, and extended chains range from 11 to 18%. Random curls and α-helices constitute the predominant secondary structures of the MsPYL proteins, while extended chains are dispersed throughout the entire protein.

GO and KEGG enrichment analysis of the MsPYL gene

GO analysis revealed that 41 MsPYL genes were enriched markly in the molecular function (MF) categories of transcription factor activity, transcription factor binding, and protein binding (GO: 0000989, GO: 0003712, GO: 0000988). KEGG pathway analysis indicated that the MsPYL genes were primarily involved in plant hormone signal transduction (Mtr04075) and the plant MAPK signal transduction pathway (Mtr04016).

Tissue-specific expression analysis of the MsPYL gene

Through an in-depth analysis of the sequences of 41 MsPYL genes, it was determined that the upstream promoter sequences predominantly contained response elements related to hormones, light, low temperature, stress, and growth. Notably, hormone and light response elements comprised the largest proportion within MsPYL genes (Fig. 12). Therefore, the expression characteristics of the MsPYL genes in roots and leaves were investigated by applying various hormones. MsPYL41, MsPYL40, MsPYL39, and MsPYL29 were expressed in the roots throughout the entire growth period. The expression levels of these genes increased with the duration of hormone treatment, peaking at 12 h. Notably, they were significantly induced by ABA, indicating that these genes not only contribute to the normal regulation of plant growth but also play a role in stress resistance. The expression levels of MsPYL8, MsPYL19, MsPYL4, and MsPYL11 increased with the duration of treatment, and they were exclusively expressed in response to hormonal stress. Especially between 6 and 12 h, IAA, ABA, and SA greatly induced them, with MsPYL4 exhibiting the most pronounced induction effect of ABA at the 12 h mark. In contrast, MsPYL41, MsPYL40, MsPYL39, and MsPYL29 were greatly induced by PEG, SA, and ABA from 6 h to 24 h; however, their expression levels were notably lower than those observed in the roots.

Discussion

ABA has attracted wide attention as an indispensable factor in regulating plant growth and responding to abiotic and biotic stresses [34–35]. ABA in plants is sensed by ABA receptors Pyrabactin resistance 1 (PYR1) and PYR1-like (PYL) proteins (PYLs), which activate downstream signaling regulatory networks [36]. Promoting the binding of downstream genes is the initial step of ABA signaling pathway and plays a crucial role in plant physiological and metabolic activities [37–38]. 41 MsPYL genes were identified in the alfalfa gene database by utilizing the PYL proteins sequences from A. thaliana, O.sativa, and M.truncatula in this research. Phylogenetic analysis and conserved motif analysis revealed that 41 MsPYL genes were categorized into 6 groups, each containing 6 to 9 conserved motifs with lengths ranging from approximately 6 to 15 amino acids, and most MsPYL genes exhibited three conserved motifs. MsPYL genes located within the same branch of the phylogenetic tree exhibit similar conserved motifs, as well as comparable motif structures and secondary structures at the same node [39]. Therefore, it is hypothesized that this group of genes may have similar regulatory functions. AtPYL8 exhibits high homology with MsPYL2, MsPYL4, and MsPYL8, suggesting that it may have similar regulatory functions. Additionally, AtPYL8 is associated with the growth and development of lateral roots [23, 40–41], so it is speculated that MsPYL2, MsPYL4, and MsPYL8 may also play a regulatory role in the development of lateral roots in plants [42].

AtPYL1 and AtPYL3 have been shown to regulate ABA-induced stomatal closure and enhance water use efficiency [23], MsPYL1 and MsPYL3 exhibit high homology with AtPYL1 and AtPYL3, which indicates that these genes may also have the biological function of regulating stomatal closure [9]. The PYL gene family members of A. thaliana, O.sativa, M.truncatula, and M.sativa appear to belong to the same evolutionary branch (Group V) based on gene evolution analysis, suggesting that they may share similar evolutionary trajectories [41, 43]. In addition, the molecular weights and isoelectric points of 41 PYL proteins are different, which indicates that they may function in distinct microenvironments. The MsPYL genes were primarily located in the cytoplasm according to subcellular localization predictions [44], which aligns with findings in A. thaliana, O.sativa, and other plant species [45]. This indicates that the PYL genes are conserved across different plant species. Through KEGG and GO enrichment analyses, 41 MsPYL genes were enriched markly in the plant hormone signaling pathway, the plant MAPK signaling pathway, transcription factor activity, transcription factor binding, and protein binding, which showed that MsPYL may play a regulatory role in various biological processes [46].

It has been observed that when plants experience stress, they produce corresponding resistance factors through a series of signal transductions. This process regulates the cis-acting elements upstream of related genes, initiating the expression of stress-related genes [47]. Ultimately, plants respond to stress through phenotypic changes and physiological metabolic activities [48]. It was discovered that the promoter region of MsPYL contains numerous cis-elements, primarily associated with hormones, light, low temperatures, stress, and growth and development [49]. It is speculated that the MsPYL proteins not only play a role in regulating growth and stress resistance, but that their gene expression is also influenced by various environmental factors. Hormones and light-responsive elements constitute the largest proportion, indicating that the regulatory expression of the MsPYL genes may be influenced by light and hormones during the growth and development of alfalfa. Plant hormone signals play a crucial role in the overall growth and development of plants, as well as in their ability to withstand various stresses [50]. Alfalfa contains a great amount of MeJA, ABA, SA, and other hormone-response elements [51–52], indicating that the expression of MsPYLs during stress is closely related to various hormones [53].

PYL gene family is not only important but also versatile in plant growth and development. In A. thaliana and O.sativa, AtPYL1, AtPYL2, AtPYL4, AtPYL5, AtPYL8, AtPYL13, OsPYL5, and OsPYL6 play crucial roles in regulating seed germination, stomatal closure, and the expression of genes related to the ABA signaling pathway [13, 54,55,56,57]. The expression of AtPYL5 and AtPYL8 may influence the cold resistance of plants [58]. Additionally, AtPYL8 can enhance the transcription of auxin-related genes, thereby regulating the growth of lateral roots [9]. Furthermore, MsPYL4 and AtPYL8 exhibit high homology and may share similar regulatory functions. In this study, MsPYL3 and MsPYL8 exhibit high homology with AtPYL3 and AtPYL8, suggesting their potential role in controlling stomatal closure, enhancing drought resistance, and regulating lateral root growth in response to stress [59]. The genes MsPYL2, MsPYL4, MsPYL8, and AtPYL8 were identified as belonging to the VIa group, indicating a close evolutionary relationship. It is proposed that the MsPYL2, MsPYL4, and MsPYL8 genes play a significant role in regulating lateral root growth and drought resistance during the growth of alfalfa [17, 60].

Conclusion

41 MsPYLs genes were identified from the reference genome of alfalfa, which are unevenly distributed across 24 chromosomes in this study. MsPYL proteins are relatively small, with a sequence length ranging from 175 to 231 amino acids and a molecular weight between 19.59 and 25.31 kDa. MsPYLs can be categorized into 8 groups based on phylogenetic relationships, conserved motifs, and gene structures, all of which contain PYR/PYL/RCAR domains. MsPYLs members contain cis-acting elements that are involved in plant growth and development, as well as hormone and stress responses. Transcriptome data revealed that different MsPYL genes may play a crucial role in the growth and development of alfalfa, as well as in its response to drought stress. qRT-PCR analysis demonstrated that the PYL gene can respond to various stimuli, including drought, ABA, SA, GA and IAA. Furthermore, the gene is expressed in rhizomes and leaves to varying degrees, indicating a close relationship between the PYL gene and stress response. The results of this study provide a theoretical basis for further exploration of the function of the MsPYLs gene and offer new candidate genes for the improvement and innovation of drought-resistant alfalfa germplasm.

Fig. 1
figure 1

The working model of PYL-dependent ABA binding and inhibition of PP2C activity

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Fig. 2
figure 2

Chromosome distribution of MsPYL genes family members in alfalfa

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Fig. 3
figure 3

The evolutionary tree of MsPYL genes family

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Fig. 4
figure 4

The gene structure and motif distribution of the MsPYL genes

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Fig. 5
figure 5

Cis-acting elements in the promoters of each MsPYL genes

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Fig. 6
figure 6

Schematic diagram of the syntenic relationships of MsPYL gene family in alfalfa. Different colored lines indicate the collinearity between two genes

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Fig. 7
figure 7

Collinearity between Medicago. sativa, M. truncatula and A. thaliana MsPYL genes. The red line indicates collinearity between two genes across different species

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Fig. 8
figure 8

Domain information of the MsPYL genes family

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Fig. 9
figure 9

Heatmap showing the expression of members of the MsPYL genes family under drought stress

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Fig. 10
figure 10

Gene structure analysis of MsPYL genes family

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Fig. 11
figure 11

Secondary structure analysis of MsPYL genes family

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Fig. 12
figure 12

MsPYL genes tissue specificity analysis

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Data availability

Sequence data that support the findings of this study have been deposited in the European Nucleotide Archive with the primary accession code PRJNA1098431.

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Funding

This work was supported by the National Natural Science Foundation of China (No. 32160327), the China Forage and Grass Research System (No. CARS‑34), the Excellent Doctoral Student Program (24JRRA675) and the “Innovation Star” project for outstanding postgraduates in Gansu Province (2023CXZX‑624) and (2025CXZX-841).

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  1. Kun Wang and Jiao Cheng contributed equally to this work.

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  1. Key Laboratory of Grassland Ecosystem of Ministry of Education, College of Pratacultural Science, Gansu Agricultural University, Lanzhou, 730070, China

    Kun Wang, Jiao Cheng, Jing-Ru Chen, Yan-Yan Luo, Yu-Heng Yao & Li-Li Nan

  2. Key Laboratory of Forage Germplasm Innovation and New Variety Breeding of Ministry of Agriculture and Rural Affairs (Co-sponsored by the Ministry and Gansu Province), Lanzhou, 730070, China

    Kun Wang, Jiao Cheng, Jing-Ru Chen, Yan-Yan Luo, Yu-Heng Yao & Li-Li Nan

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L-LN planned and designed the research. KW analyzed the data, wrote, and revised the manuscript. JC, Y-YL, J-RC, and Y-HY made plentiful valuable comments for the manuscript. All authors read and approved the final manuscript.

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Wang, K., Cheng, J., Chen, JR. et al. Genome-wide identification of pyrabactin resistance 1-like (PYL) gene family under phytohormones and drought stresses in alfalfa (Medicago sativa). BMC Genomics 26, 383 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11575-0

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BMC Genomics

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