Comprehensive analysis of potato (Solanum tuberosum) PYL genes highlights their role in stress responses

Genome-wide identification of PYL gene family in potato

We obtained the protein sequences of 14 AtPYLs from The Arabidopsis Information Resource (TAIR) website (https://www.arabidopsis.org). We used the Spud DB Potato Genomics Resources website (http://spuddb.uga.edu) to obtain the genomic and protein datasets of potato (Solanum tuberosum L.). For the four other plant species (Oryza sativa L., Capsicum annum L., Solanum lycopersicum L., and Zea mays L.), we obtained the data from the ensemble plants website (http://plants.ensembl.org/index.html). Finally, to search the PYLs in protein datasets, 14 AtPYL proteins were used as query with local BLASTP search (Altschul et al. 1997) using threshold e-value 1e⁻⁵ with and 50% minimal alignment coverage (Shahzad et al. 2023b). Moreover, in order to verify the presence of Polyketide_cyc2 domain in the obtained candidate proteins, firstly we used the Pfamscan website (https://www.ebi.ac.uk/Tools/pfa/pfamscan/; see Supplementary Table S1) then both the START and NCBI CD-search tools used to further verify the domain presence (Letunic et al. 2021; Wang et al. 2023).

Prediction of physicochemical characteristics and subcellular localisation

To examine the physicochemical characteristics of StPYL proteins, we used the ExPasy server (https://web.expasy.org/protparam/). We estimated the number of amino acids, protein’s molecular weights, theoretical isoelectric points, protein instability index, and the overall hydropathicity (GRAVY). Additionally, we used the Plant-mPLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) to predict the subcellular localisation of the StPYL proteins (Chou and Shen 2010).

Phylogenetic analysis, gene structure, and conserved motif analysis

The protein sequences of PYLs from Arabidopsis thaliana L., S. tubersoum, S. lycopersicum, C. annuum, O. sativa, and Z. mays were used for phylogenetic analysis to elaborate the evolutionary relationship of potato’s PYL gene with other species. To construct the phylogenetic tree, we used the MEGA X software (Kumar et al. 2018). Initially, the protein sequences were aligned using ClusterW with default parameters and then phylogenetic tree was created by using neighbour-joining (NJ) method (Shahzad et al. 2023b) with the following parameters: bootstrap values with 1000 replicates, using the Jones-Taylor-Thornton (JTT) + invariant sites (I) + Gamma (G) substitution model (Kumar et al. 2018). Finally, the resulted tree was visualised using the online tool Interaction Tree of Life (iTOL) (Letunic and Bork 2021).

Gene structure of StPYLs were examined by the Gene Structure Display Server 2.0 (GSDS 2.0; https://gsds.gao-lab.org/). The exons and introns of all StPYLs were identified and then visualised by the TBtools program. We used the Multiple Expectation Maximisation for Motif Elucidation (MEME, ver. 5.5.3) online tool with default settings to find conserved motifs in the StPYLs.

Chromosomal location and synteny analysis

The chromosomal locations of all StPYLs were determined using the positional information from the annotation of the potato genome sequence. Using the TBtools mapping of the PYL genes to the relevant chromosomes, we were able to see where the PYL genes were located within the genome. We investigated the gene duplication within the PYL gene family using TBtools with default parameters. They were located based on the precise positions of the tandemly duplicated genes on each chromosome. To assess the level of selective pressure on the dataset, we computed the synonymous (Ks) and non-synonymous (Ka) replacement rates as well as the Ka/Ks ratio.

To provide a full understanding of the synteny relationship between orthologous PYL genes, we constructed syntenic analysis maps using the default parameters of TBtools Multiple Synteny Plotter. This study identified conserved genomic regions and the degree of synteny among the PYL genes. The homologous genetic linkages of the PYL genes were visualised using the TBtools Amazing Super Circos program.

Evaluation of cis-acting elements in StPYL promoters

To identify the cis-acting elements, up to 2000 bp upstream of the initiation codon (ATG) of 17 StPYLs were extracted using TBtools. Then all of the 2000 bp sequences were uploaded to the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for the investigation of plant cis-regulatory components of all StPYLs (Conesa et al. 2005).

Protein–protein interaction prediction analysis and miRNA target prediction

Interaction networks of StPYL proteins were predicted using the STRING database (http://string-db.org/cgi) and displayed through Cytoscape software (ver. 3.8.2). The targeted relationship between microRNA (miRNA) and PYL genes in potato was predicted with psRNATarget (https://www.zhaolab.org/psRNATarget/) and then visualised by Cytoscape (ver. 3.8.2).

Expression profile of StPYLs in different tissues and under various stresses

To examine the expression patterns of 17 StPYL genes, we used the Potato Genome Sequencing Consortium’s website (http://spuddb.uga.edu/pgsc_download.shtml) for data of FPKM (fragments per kilobase of transcript per million mapped reads) extraction. Comprehensive expression data for representative transcripts from the 40 DM and 16 RH libraries were provided in this dataset.

Fourteen different potato tissues were studied for StPYL expression, including the callus, roots, shoots, leaves, petioles, stolons, tubers, stamens, sepals, carpels, petals, full flowers, immature fruits, and mature fruits. This dataset also included the expression of gene under various biotic (P. infestans, β-aminobutyric acid (BABA), benzothiadiazole (BTH) treatments under stress conditions) and abiotic (salinity, drought, heat) stresses. To display the expression patterns, heatmaps of StPYLs expressions were made using the TBtools (Chen et al. 2020a). Heatmaps were created to display the variation in StPYL gene expression levels across different tissues and developmental stages, and under different stresses using the normalised log₂(FPKM) values.

Plant material and stress treatments

The tetraploid potato cultivar Desiree was utilised as the experimental subject in this investigation. Aseptic explants were generated from 4-week-old sterile seedlings of Desiree cultivated in culture vessels. These explants were subsequently sub-cultured on 30 mL of Murashige and Skoog (MS) medium for a duration of 30 days. Following this, potato seedlings were transplanted into pots and placed in greenhouses under a photoperiod of 16 h of light and 8 h of darkness at a temperature of 22°C. To analyse the expression of the PYL genes, abiotic stresses including 150 mM NaCl (salinity) and 260 μM mannitol (dehydration) were applied to whole plants for durations of 0, 5, and 10 h. Biotic stress treatments involved the imposition of P. infestans on the leaves for 10 h. Samples were collected at 0, 5, and 10 h post-treatment.

Total RNA extraction and reverse transcription quantitative PCR (RT-qPCR)

Total RNA was extracted from various samples using the SteadyPure Plant RNA Extraction Kit (Accurate Biology, Changsha, China). The extracted RNA was subjected to a purity assay using a NanoDrop 2000c spectrophotometer (Thermo, USA), where the OD260/OD280 ratio was employed to evaluate the level of protein contamination, with readings within the range of 1.8–2.1 indicating high-quality RNA. RNA integrity was further assessed by loading samples onto a 1.0% agarose gel with 1× TAE running buffer for electrophoresis, ensuring that the RNA was intact before proceeding to complementary DNA (cDNA) synthesis. Subsequently, cDNA was synthesised using the PrimeScript RT Reagent Kit (TakaRa, Kusatsu, Japan) following the manufacturer’s instructions. RT-qPCR was conducted with the following program: (1) preincubation at 95°C for 2 min; (2) denaturation at 95°C for 15 s; and (3) annealing/extension at 60°C for 20 s, repeated for 40 cycles. The remaining steps were performed according to the default settings of the instrument. The EF1a gene served as the reference gene. Three independent biological replicates were analysed, and gene expression levels were quantified using the 2^−ΔΔCt method. The primer sequences for RT-qPCR are provided in Table S2. Finally, the data visualised using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Data were presented as mean ± s.e. Statistical significance was assessed using Student’s t-tests (*P < 0.05; **P < 0.01).

Identification and features of StPYLs

We used 14 AtPYL protein sequences as references to identify the PYL protein in potato genome and identified the 17 StPYL proteins that were named according to their physical locations on the corresponding chromosomes (Table 1). Further, we investigated the physiochemical properties of StPYLs. The amino acid length varied among StPYLs ranging from 154 (StPYL6) to 231 (StPYL11). The relative molecular weight ranged from 17,274.54 Da (StPYL6) to 25,285.95 Da (StPYL11) (Table 1). Based on the isoelectric points (pI), 11 StPYLs proteins were acidic nature with pIs ≤ 6.5, five were neutral 6.5 < pI < 7.5, and only one was alkaline with with pIs > 7.5. (Table 1). Meanwhile, the instability index indicated about the stable and unstable StPYL proteins while the GRAVY values indicating their hydrophilic nature. The subcellular localisation analysis predicted that most StPYL proteins were primarily localised in cytoplasm with few exceptions to mitochondrion, nucleus, and chloroplast.

Mapping of the PYL gene loci onto the potato chromosomes revealed that the 17 StPYLs distributed unevenly across nine chromosomes (Fig. 1). Notably, StPYL3/4/5/6 were clustered on chromosome 3 (Chr 3), while StPYL10/11/12 were located on Chr 8. Furthermore, single PYL genes were identified on Chr 1, Chr 2, Chr 5, and Chr 9 (StPYL1, StPYL2, StPYL7, and StPYL13, respectively). Dispersion of StPYL genes across multiple chromosomes could suggest functional divergence or redundancy within the gene family, allowing for fine-tuning of ABA signalling in various tissues or developmental stages.

Fig. 1.

Chromosomal distribution of PYL genes in the Solanum tuberosum genome. Chromosomal information for the StPYL genes was obtained from the S. tuberosum genome annotation file and TBtools was used to display this distribution.

Phylogenetic and molecular evolution analysis of StPYLs

To examine the evolutionary relationship of potato PYL proteins, we created a phylogenetic tree by using the protein sequence alignment of 17 StPYLs along with 14 AtPYLs, 12 OsPYLs, 11 CaPYLs, 14 SlPYLs, and 14 ZmPYLs (Fig. 2). These proteins were divided into three subfamilies: (1) Subfamily I with 31 PYLs; (2) Subfamily II with 20 PYLs; and (3) Subfamily III with 31 PYLs (Fig. 2). Representatives from both eudicots and monocots in all three groups indicate that PYLs originated before their divergence, suggesting long-term conservation of PYL genes evolution across plant lineages.

Fig. 2.

Phylogenetic analysis of PYL gene family from Solanum tubersoum, Arabidopsis thaliana, Solanum lycopersicum, Capsicum annuum, Oryza sativa, and Zea mays. Phylogenetic tree was constructed by MEGA X using neighbour-joining method with 1000 bootstrap values. PYL proteins are divided into three subfamilies: (1) I, green; (2) II, turquoise; and (3) III, pink.

Gene structure and conserved motifs of StPYLs

To elucidate the organisation of StPYLs, we thoroughly examined the exon-intron architecture of these genes and further analysed the conserved motifs in their protein sequences (Fig. 3). Gene structure results revelled that the three genes from Subfamily I contain the two introns, one contains three introns while one gene contain no introns. All the genes from Subfamily I contain two exons except StPYL2 that has only one exon. In Subfamily II two genes (StPYL17, StPYL10) contain two introns and two exons, while other two genes have only single exon. Subfamily III members contain a single exon and lack introns.

Fig. 3.

PYL gene structure and conserved motifs analysis. (a) Phylogenetic tree of StPYLs. Subfamily I, green boxes; Subfamily II, turquoise boxes; Subfamily III, pink boxes. (b) Conserved motifs were detected using MEME and are represented by boxes of different colours. (c) Gene structures were generated using the Gene Structure Display Server (GSDS 2.0), exons (CDS) and introns are indicated with green ellipses and black lines, respectively, and yellow ellipses indicate untranslated regions (UTRs).

Using the MEME website, we discovered 10 conserved motifs in StPYLs (Fig. 3b, Table S3). Among these, three motifs (Motif 1, Motif 2, and Motif 3) were found to have known descriptions: ‘Pathogenesis-related protein Bet v 1 family’, ‘Polyketide cyclase/dehydrase and lipid transport’, and ‘BDHCT (NUC031) domain’, and these three motifs were present in all members of StPYLs, which provides insight into the functional roles of the PYL proteins. While some motifs were specific to the subfamilies. Subfamily I members contained Motif 8 and Motif 9 with some exceptions such as StPYL2 did not contain these motifs. The members of Subfamily II contained Motif 6 while Subfamily III members contained Motif 4, Motif 5, and Motif 7. The remaining seven motifs, although not having explicit descriptions, likely play essential roles in the structural integrity and function of PYL proteins. These conserved motifs may be crucial for the proper folding, stability, and interaction of PYL proteins with other components of the ABA signalling pathway. Understanding the precise roles of motifs requires further investigation, possibly through structural and functional analyses. These findings suggest that proteins within the same subfamily possess motifs with similar properties, indicating potential shared biological roles. In contrast, proteins with distinct motifs may perform specialised or unique functions.

Gene duplication and synteny analysis of StPYLs

Gene family formation and evolution are significantly influenced by tandem, segmental, and whole-genome duplication. Our findings revealed the gene duplications events. We found the 10 gene duplication events in potato PYL gene family through segmental duplication (SD). These duplicated genes were distributed across Chr 1, Chr 3, Chr 5, Chr 6, Chr 8, Chr 9, Chr 10, and Chr 12 (Fig. 4a). Further, we analysed the substitution rates of synonymous (Ka) and non-synonymous (Ks) mutations to determine the selection pressure present during duplication events. The Ka/Ks values of duplicated StPYLs ranged from 0.0560 to 0.1750, indicating that purifying selection has predominantly driven their evolution (Table 2). To explore evolutionary relationships and gene arrangement similarities across species, we conducted a synteny analysis (Fig. 4b, Table S4). We compared the synteny of StPYLs in potatoes with orthologous genes in five other plant species: two monocots (rice and maize) and three dicots (Arabidopsis, pepper, and tomato). The synteny between potato PYLs and dicots was generally greater than that with monocots. There were 13, 13, 15, seven, and five StPYLs that had collinearity with the PYLs of A. thaliana, C. annuum, S. lycopersicum, O. sativa, and Z. mays, respectively.

Fig. 4.

Gene duplication and synteny analysis. (a) Duplicate gene pairs in Solanum tuberosum. (b) Synteny between StPYLs and PYLs from five other species (Arabidopsis thaliana, Solanum lycopersicum, Capsicum annuum, Oryza sativa, Zea mays). The grey lines represent background which indicated the collinear blocks within the genomes of S. tuberosum and other species, PYL pairs were marked by red lines.

Promoter analysis of StPYLs

We examined the cis-acting regulatory elements (CREs) in the promoter regions of StPYLs to understand gene regulation. We analysed the 2000 bp upstream sequences using PlantCARE. The StPYL promoters were found to contain numerous regulatory elements, with a significant emphasis on hormone and stress-related CREs (Fig. 5). Key stress-responsive elements identified included MBS, LTR (low temperature responsive), TC-rich repeats, WUN-motif, and W box, which were present in the promoter regions of 14 StPYLs (Table S5). Notably, LTR elements were the most prevalent. Hormone-responsive elements were also abundant, with ABA response elements (ABRE) and ethylene responsive element (ERE) being the most prominent. Other hormone-related elements including P-box, TGACG-motif, CGTCA-motif, TCA-element, TATC-box, GARE-motif, and TGA-element, indicating responsiveness to various hormones such as ABA, ethylene, auxin, and gibberellins. The presence of multiple auxin-responsive and gibberellin-responsive elements in the promoters suggests a potential interaction between phytohormones in regulating ABA receptor development. StPYL1 exhibited the highest number of cis-acting elements, followed by StPYL2, StPYL2, StPYL6, and StPYL8. Except StPYL3/5/4, all other StPYLs contained LTR and TC-rich repeats within their promoter regions. While this analysis provides a comprehensive overview of CREs, further experimental validation in necessary to confirm their functional relevance. Previous studies have shown that similar CREs play crucial roles in gene regulation under stress conditions and hormone treatments, which support our findings (Billah et al. 2022; Liu et al. 2022; Marand et al. 2023).

Fig. 5.

Genomic distribution of cis-regulatory elements in StPYL promoter regions. Different cis-regulatory elements are indicated with different colours. The cis-regulatory elements in promoter regions were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) and drawn by TBtools.

Protein interaction networks analysis of StPYLs and miRNA targets

The STRING database was used to predict the protein–protein interactions (PPI) of the StPYLs, which enhanced our understanding of the biological function and regulatory network associated with them. The results showed the 14 StPYLs interacted with 21 functional proteins.

Due to PYL protein involvement in ABA signalling their interacting proteins were expected to be involved in ABA signalling complex. As anticipated, the majority of the proteins were predicted to interact were crucial and functionally verified constituents of the ABA signalling complex, such as clade A protein phosphatase 2C (PP2C) and SNF1-related protein kinase 2 (SnRK2), as shown in Fig. 6. Moreover, we observed that PYL proteins may have interactions with polyketide synthases (PKSs). According to functional annotations, these interacting proteins of StPYLs can be divided into three groups: (1) five phosphatase 2C family proteins; (2) seven serine/threonine-protein kinases; and (3) three protein phosphatase 2C-like proteins. Additionally, one MYB27 protein showed an indirect interaction with StPYL proteins (Fig. 6). Although our analysis offers predictions, experimental validation will be necessary in future studies to thoroughly explore the relevance of these findings.

Fig. 6.

Predicted protein–protein interaction networks of StPYL proteins with other potato proteins using STRING tool. The green circles represent potato PYL proteins, and the circles on the inside represent proteins that interact with StPYLs. The two circles connected by the grey line represent the interaction between the proteins.

Moreover, the relationships between the miRNAs and StPYLs were predicted using psRNATarget database. We identified that all 17 StPYLs were targeted by several miRNAs. StPYL15 were targeted by 20 miRNAs, which is the most targeted gene. StPYL6 and 16 were targeted by six miRNAs, while StPYL12 were targeted by five miRNAs, respectively. StPYL1, StPYL8, and StPYL10 were targeted by three miRNAs while StPYL13 were targeted by only one miRNA. Each of the other 10 StPYLs was targeted by two miRNAs (Fig. 7).

Fig. 7.

miRNA targets of PYL genes in Solanum tuberosum. Geen circles, miRNAs; pink circles, StPYL genes.

Expression pattern of StPYLs among different potato tissues

To investigate the biological functions of StPYL gene, we examined the publicly available transcriptome data set 14 different tissues, including callus, leaves, roots, shoots, tubers, sepals, petals, stamens, mature flowers, petioles, carpels, stolons, mature fruit, and immature fruit, and we found the tissue-specific expression patters of StPYLs. For example, StYPL5/4/9/13/14/15 exhibited relatively higher expression levels in stolons, indicating their potential importance in potato seed development (Fig. 8). Additionally, StYPL3/4/5/7/8 showed notable expression levels in callus and tubers, suggesting their significance in these tissues. StPYL10 and StPYL17 displayed substantial expression levels in shoots. StPYL11 exhibited exclusive and high-level expression in petioles, while StPYL2, StPYL11, and StPYL12 showed relatively lower expression levels across various tissues (Fig. 8). However, StPYL2 exhibited high expression level in mature fruits than the other tissues. These variations in the expressions of StPYLs across different tissues indicate their involvements in diverse stages of potato growth and development, potentially contributing to geographical differences in potato phenotypes.

Fig. 8.

Expression profile of StPYLs in 14 tissues based on transcriptomic data. The heatmap was generated using the log₂ expression levels (FPKM). The colour bar to the right represents the log₂ expression value, with red representing upregulated, and blue representing downregulated expression level. PYL Subfamily I, green; Subfamily II, turquoise; and Subfamily III, pink.

Response of StPYLs expression to various stresses

We examined the expression patterns of the 17 StPYLs under various abiotic and biotic stresses (Figs 8 and 9). Under abiotic stresses, we observed the diverse expression pattern such as StPYL6 showed the higher expression level under drought and salt stress, while StPYL1 showed higher expression level under all observed abiotic stress. StPYL11/13/14/15 showed the lower expression pattern under drought and salt stresses, while StPYL13/14 showed the upregulation under heat stress (Fig. 8). For the biotic stress the results showed that among StPYLs, five members of StPYL3/5/4/8/13, exhibited high expression exclusively in response to P. infestans, indicating their potential roles in this specific pathogen-induced stress. In contrast, five other members StPYL6/12/15/14/8 showed relatively lower expression levels in response to BTH and BABA treatments, suggesting their involvements in different aspects of biotic stress response pathways (Fig. 9). Notably, StPYL2 and StPYL13 demonstrated high expression specifically in response to BABA treatment, indicating their unique role in this particular stress condition. By examining the expression of StPYLs under diverse biotic stress conditions, we gain insights into their potential roles in specific signalling pathways activated during biotic stress responses.

Fig. 9.

StPYL genes expression levels in response to various biotic and abiotic stresses. The heatmap was generated using the log₂ expression levels (FPKM). The colour bar to the right represents the log₂ expression value, with red representing upregulated and blue representing downregulated expression level. PYL Subfamily I, green; Subfamily II, turquoise; and Subfamily III, pink. Yellow bars, abiotic stresses; green bars, biotic stresses.

Expression validation of selected StPYLs under various stress treatments

Further to validate the expression pattern of StPYLs under the abiotic and biotic stresses, we selected the eight StPYL genes and performed RT-qPCR to access their expression pattern under drought, salt (abiotic stresses), and P. infestans (biotic stress) stresses. We accessed the expression level at three time intervals (0, 5, and 10 h) and we observed the significant variation (Fig. 10). Specifically, StPYL6 showed the upregulation under all the treatments and the expression level increased with time interval in drought and P. infestans stresses while it decreases in salt stress. All the observed genes showed the upregulation in their expression level under P. infestans stress except StPYL11, which showed downregulation and this downregulation increased with the time interval. However, the observed genes showed the downregulation of their transcript level under the drought and salt stresses and their downregulation varied with the time interval in some genes.

Fig. 10.

RT-qPCR analyses of differential expression of eight StPYL genes under drought, salt, and Phytophthora infestans stresses. Asterisks indicate a statistically significant difference (*P < 0.05, **P < 0.01, Student’s t-test).