Genome-wide identification and expression analysis of the cucumber PYL gene family

Plant materials and treatment

The germinated seeds (Cucumis sativus L, “L306” cultivar) were treated by 5% NaClO 10 min and deionized water washing 3–5 times. After that, the cucumber seeds were put into an artificial climate incubator to promote germination. The cultivation condition: relative humidity 80%, temperature 25 °C/18 °C (day/night), and the light intensity during the day is 250 µmol m⁻² s⁻¹. After 4 days of culture, cucumber seedlings were soaked in Yamazaki nutrient solution for hydroponics. Cucumber seedlings were treated when they grew to three true leaves. This experiment set up four treatments (T1: 50 µmol/L ABA, T2: 100 μmol/L ABA, T3: 200 mmol/L NaCl, T4: 10% PEG). The cucumber leaves were collected after 0 h, 3 h, 6 h, 12 h and 24 h. Finally, put fresh samples in liquid nitrogen, immediately and stored in −80 °C refrigerator.

Genome-wide identification of cucumber PYL gene family

The protein sequences of 14 PYL genes in Arabidopsis were downloaded from the Arabidopsis information resource (TAIR) website (https://www.arabidopsis.org/). And then, the BLASTp comparison was carried out in cucumber genome data (http://cucurbitgenomics.org/), the retrieval threshold was set as E-value < E⁻¹⁰ (Altschul et al., 1997). Scaffold assemblies of three cucumber lines (Chinese long ‘9930’, Gy14, and B10) are available so far. This draft genome was assembled using Chinese long inbred line ‘9930’ version 3 (Cavagnaro et al., 2010). The login number of duplicate genes in the comparison results was manually deleted. Then the gene domain after screening was searched in the Pfam database (http://pfam.xfam.org/search#tabview=tab1). A total of 15 CsPYL genes were obtained. The Polyketide_cyc2 domains (PF10604) were found in all CsPYLs. After naming according to the chromosome order, it was found that CsPYL1 had an unknown domain and a large molecular weight, so it was removed. Then downloading HMM files for HMM search comparison, a total of 14 PYL genes family members in cucumber were identified. Finally, through NCBI-CDD database (https://www.ncbi.nlm.nih.gov/cdd/) (Marchler-Bauer et al., 2015) identified whether the candidate sequences have PYR/PYL (RCAR) like domain (cd07821).

Analysis of chromosome location and collinearity analysis

The Chinese long ‘9930’ V3 version of gff3 file was downloaded from cucumber genome database, and the chromosome location and chromosome length information of 14 PYL genes were screened out and named CsPYLs according to their distribution on chromosomes. The MG2C (http://mg2c.iask.in/mg2c_v2.0/) was used to map the chromosomal location. Using Mcscanx search for homology, the protein-coding genes from cucumber genome was compared against itself and Arabidopsis genomes using BLASTp and retrieval threshold was set as E-value < E⁻⁵. Others were not modified by default parameters. Whole-genome BLASTP results were used to compute collinear blocks for all possible pairs of chromosomes and scaffolds (Wang et al. 2012a). Subsequently, TBtools was used to highlight the identified PYL collinear pairs and their collinear pairs with Arabidopsis (Chen et al., 2020).

Selective pressure analysis of cucumber PYL gene family

PAL2NAL (http://www.bork.embl.de/pal2nal/index.cgi?) were used to calculate the non-synonymous/synonymous (d_N/d_s) value of duplicate gene pairs (Goldman & Yang, 1994).

Sequence analysis and basic information of cucumber PYL gene family

The subcellular localization of PYL protein in cucumber was predicted by Wolf PSORT (https://wolfpsort.hgc.jp/) (Xiong et al., 2015). The number of amino acids, isoelectric point (PI), molecular weight (MW) and other physical and chemical information of CsPYL genes were identified on the ExPASy website (https://web.expasy.org/protparam/) (Artimo et al., 2012). Additionally, the secondary structure of cucumber PYL protein were predicted through the online web site (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html).

Construction of phylogenetic tree

The phylogenetic tree of PYL gene family of cucumber, A. thaliana, rice, soybean, grape, B. distachyon and apple were constructed by using MEGA 7 software with muscle methods to align multiple sequences (Kumar, Stecher & Tamura, 2016). The amino acid substitution model was (JTT+G) (Supplemental Information 3), and the bootstrap values were calculated for 1,000 replicates. Finally, a stable minimum neighbor tree was selected to represent their evolutionary relationship and beautify the constructed tree by ITOL website (Ivica & Peer, 2016) (http://itol.embl.de/).

Analysis of gene exon-intron structures and protein conserved motifs

Upload CDS and genome-wide sequences of 14 cucumber PYL genes on GSDS (GSDS v2.0) websites (http://gsds.gao-lab.org/) (Yang et al., 2015) for genetic structure analysis; The conserved motifs of 14 cucumber PYL genes were analyzed by MEME website (http://meme-suite.org/tools/meme) (Bailey et al., 2009). The maximum motif number was set to 10, and the remaining parameters were the default values. Functional annotations of these motifs were performed using HHpred (https://toolkit.tuebingen.mpg.de/tools/hhpred) (Zimmermann et al., 2018).

Analysis of cis-acting elements in CsPYL gene promoters

In order to predict the cis-acting elements in the promoter of CsPYL genes, TBtools was used to extract the genomic sequences of 14 cucumber PYL genes up to 2,000 bp upstream of the initiation codon (ATG). Then, the 2,000 bp sequences of 14 CsPYLs were submitted to the online website plantcare for prediction (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Lescot, 2002).

Transcriptome analysis of CsPYL genes in different tissues

In order to study the gene-specific expression of CsPYL genes in different tissues of cucumber, the accession number PRJNA80169 was used from cucumber genome data (http://cucurbitgenomics.org/) to obtain cucumber RNA samples from different tissues and organs (tendril-base, tendril, root, leaf, stems, ovary-unfertilized, ovary-fertilized, ovary) RNA-Seq data (Li et al., 2011; Harris et al., 2008). Finally, the method of log2 was used for data conversion and Tbtools software was used to draw the expression heat map of CsPYL gene.

RNA extraction and real-time RT-PCR

Total RNA was isolated from collected samples using a Plant RNA Extraction kit (Tiangen, China). The complementary DNA was synthesized by using fastking cDNA dispersing RT supermaxs kit (Tiangen, China) with 2 µl RNA as template. The CDS sequences of CsPYL genes were input into the homepage of Shanghai biology company (Shanghai, China) for online primer design (Table S1), and then the primer sequences were synthesized. The SYBR Green kit (Tiangen, China) was used for fluorescence quantitative analysis. The volume of reaction system was 20 µL, which contained 2 µL cDNA solution, 10 µL2^∗SuperReal PreMix Plus, 0.6 µL of 10 µM forward and reverse primers, 0.4 µL 50^∗ROX Reference Dye and 6.4 µL of distilled deionized water. Then the qRT-PCR was performed with LightCycler^® 480 II real-time fluorescence quantitative PCR instrument. The amplification program conditions were as follows: 95 °C for 15 min, and 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Each sample was replicated 3 times. The relative expressions of CsPYL genes were calculated by 2^−ΔΔCT method (Livak & Schmittgen, 2002), with CsActin as an internal control (Gan et al., 2013; Wan et al., 2010). Each sample repeat at least three times.

SPSS 20.0 was used for one-way ANOVA, and Duncan method was used to test the significance of the difference (P < 0.05) and data were all the average of 3 replicates. The Excel was used to complete the histogram of relative expressions.

Genome-wide analysis and chromosome distribution of CsPYL gene family

We determined that 14 putative CsPYL were present in the cucumber genome through BLASTP by using 14 AtPYL sequences as references. From the analysis of their physical and chemical properties (Table 1), PYL2 has the largest number of amino acids and the largest molecular weight of protein. The other members of PYL protein have 162∼233 amino acids and protein molecular weight range 18.17 from 25.66 KDa. In addition, isoelectric points (PI) are from 4.91 to 8.28. Only CsPYL4 is basic protein, and the others are acidic proteins. The total average hydrophobic index of 14 CsPYL genes family members were less than zero, all of them were hydrophilic proteins.

Subcellular localization prediction display (Table 2) that most of the PYL genes in cucumber were predicted to be located in cytoplasm and nucleus, while CsPYL6, CsPYL8, CsPYL12, CsPYL13 and CsPYL14 were located in chloroplast. The results of secondary structure prediction (Table 2) found that the secondary structure of most CsPYL proteins were mainly composed of α-helix and irregular curl, and the proportion of extended structure and β-angle were the smallest.

Moreover, to predict the position of CsPYLs gene on the chromosome information, using the online website MG2C to map the chromosomal location. The distribution of PYL genes on chromosomes showed that 14 members of PYL gene family were distributed on 6 chromosomes of cucumber (Fig. 1). There were more PYL genes on chromosomes 3 (PYL3-7) and 5 (PYL10-12). Both CsPYL1 and CsPYL2 were located on the same site of Chr 2 and belonged a pair of tandem duplication genes.

Collinearity analysis of the PYL gene family in cucumber

Using Mcscanx and TBtools to analyze the segmental duplication events, it was found that there were 5 pairs of collinearity genes in cucumber (Fig. 2A), including 4 segmental duplication events between different chromosome, containing (CsPYL4/CsPYL10, CsPYL6/CsPYL8, CsPYL8/CsPYL13, CsPYL12/CsPYL13), the other 1 duplication events within the same chromosome, including (CsPYL3/CsPYL7).

Subsequently, we also explored the collinearity relationships between the cucumber PYL genes and Arabidopsis (Fig. 2B). There were 19 collinear gene pairs between 11 CsPYLs and 11 AtPYLs. According to the collinearity relationship between CsPYLs and AtPYLs, most collinear gene pairs were distributed on At-Chr 2, 4, 5 and Cs-Chr 3-5. In addition, the collinear relationship between most CsPYLs and AtPYLs was one-to-many, such as the collinear relationships between AtPYL1 and CsPYL3, 7; AtPYL4 and CsPYL6, 8, 13. The genes with collinear relationship in our study belong to the same group in the phylogenetic tree (Fig. 3) and were very similar to their gene structure and conserved motifs (Fig. 4), such as AtPYL1, AtPYR1 and CsPYL3, 7 which all have upstream/downstream sequence and two exons (except CsPYL3).

Analysis of d_N/d_s values of PYLs in cucumber, cucumber and Arabidopsis

To further investigate the divergence and selection in duplication of PYL genes, the non-synonymous substitution rate (d_N), synonymous substitution rate (ds) and d_N/d_s values were evaluated for the homologous gene pairs among cucumber, cucumber and Arabidopsis (Table 3). When d_N/d_S > 1 is the positive selection, d_N/d _S = 1 is the neutral selection, 0 < d_N/d_S <1 is purifying selection (Yadav et al., 2015). The d_N/d_s value of all cucumber gene pairs was less than 1. Similarly, the d_N/d_s value of all collinear gene pairs in cucumber and Arabidopsis was less than 1. These data suggest that these genes were mainly under the purifying selection during evolution and could help to maintain the basic function of this gene.

Phylogenetic analysis of the CsPYLs family

In order to determine the evolutionary relationship of PYL genes, a phylogenetic tree was constructed using the 14 CsPYL genes from C. sativus, 14 AtPYLs from A. thaliana, 13 OsPYLs from O. sativa, 21 GmPYLs from G. max, 5 VvPYLs from V. vinifera, 9 BdPYLs from B. distachyon and 13 MdPYLs from M. domestica (Supplemental Information 4). A total of 89 PYL genes of the five plants were divided into three groups (Fig. 3), namely group I-III, which consisted 27, 24 and 38 members respectively. Every group contained PYLs from eudicots and monocots, hinting that these PYLs generated before the divergence from eudicots and monocots.

Gene structure and conserved domains analysis of CsPYLs

To understand the structure of the cucumber PYL gene family, we analyzed the phylogenetic relationship of the CsPYL and AtPYL proteins (Fig. 4A), gene structure (Fig. 4B) and conserved motif (Fig. 4C). The 14 CsPYL genes were divided into three groups. In the group I, all the PYL genes included one or two exons. CsPYL3, 9, 13 and 8 have two exons, and both have one intron with different lengths, among which CsPYL8 and 13 have no upstream and downstream gene sequences. In the group II, all PYL genes have three exons and two introns, only CsPYL5 has no upstream and downstream sequences. In the group III all PYL genes have one or two exons, only AtPYL13 and CsPYL11 has no non-coding regions. CsPYL12 had the longest fragment length that was more than 4,500 bp.

The protein sequences of CsPYLs and AtPYLs were analyzed by MEME website, and a total of 10 conserved motifs were identified (Fig. 4C). All the PYL members contain 2 to 5 conserved motifs. Compared with other genes, CsPYL genes in group III had no motif 1 and 2, while AtPYL8 and AtPYL5 did not have motif 3. Except all PYL genes in the group III other PYL genes have motif 4. This indicates that PYL proteins have highly conserved amino acid residues. Besides the common motif, PYL genes in each group have its own unique motif, such as AtPYL1, CsPYL7, CsPYL3, AtPYL2, CsPYL9, AtPYL3 in group I contain motif 7. CsPYL1, CsPYL2 in group III contained motif 5, motif 6 and motif 9 which were not found in other groups. From these, the proteins classified into the same group share similar motif characteristics, suggesting functional similarities among members, while presence of unique motifs might carry out unique/specialized biological functions.

Cis-Element analysis of the CsPYLs promoter in cucumber

The 2,000 bp sequence before the start codon was selected to predict cis-acting elements. As is shown in Fig. 5A, and it was found that the regulatory elements of the CsPYLs were very abundant in number and variety, mainly analyzes the hormone regulation and stress-related elements. Among the 14 CsPYLs promoters, contain 4 stress-related elements: MYC, TC-rich repeats, MBS, LTR. Additionally, there were 6 hormone-related cis-acting elements predicted, for instance, CGTCA-motif and TGACG-motif are related to MeJA responsive element; TATC-box and P-box are related to Gibberellin-responsiveness responsive element; AAGAA-motif and ABRE are related to abscisic acid response element; TCA-element are related to SA responsive element, as well as ethylene response element (ERE) and auxin response element (TGA-element).

The promoter regions of 14 CsPYL genes contain at least 6 acting elements, and all CsPYL gene contains MYC and ARE elements (except for CsPYL7), among which MYC has the largest number. It can be seen from the statistical table (Fig. 5B) that CsPYL14 has the largest number of cis-acting elements, followed by CsPYL2 and CsPYL11.

Tissue-specific expression of CsPYL genes in cucumber

In order to better understand the role of CsPYL genes in cucumber growth and development, transcriptome data from cucumber database were used to analyze their expression in different tissues. Expression patterns of 14 CsPYL genes in different tissues were constructed based on transcriptome data (Fig. 6). The expression of CsPYL1, CsPYL3-7, CsPYL10 and CsPYL14 in different eight tissues were high. On the contrary, the expression levels of CsPYL9, CsPYL11 and CsPYL13 in tissues were very low, and CsPYL13 was almost not detected, while CsPYL11 is only elevated in unfertilized ovaries. The expression of CsPYL2 was high in the fertilized ovaries, unfertilized ovaries and ovary, but even hardly detected in other parts. The spatial variations in the expression of CsPYL genes in different tissues indicate that they may participate in different stages of cucumber growth and development processes.

Response of CsPYL genes expression to various abiotic stresses and ABA treatment

PYLs, as ABA receptors, might play a vital function in abiotic stress signaling pathways. To further understand the expression of PYL genes under different abiotic stresses, qRT-PCR was used to analyze the expression of 14 cucumber CsPYL genes upon various abiotic stress treatments (including ABA, NaCl, PEG). Raw CT values were placed in (Supplemental Information 5).

As shown in Fig. 7A, under the treatment of 50 µmol/L ABA, the expression of PYL8 was up-regulated at all time points (except 24 h), and reached the highest level at 12 h, which was 30 times higher than that of 0 h (control check). Compared to the 0 h, the relative expression quantity of PYL1-3, PYL5, PYL9 and PYL10 were up-regulated about 3 h, 6 h and 12 h. However, after 24 h, the expression level of this genes was close to 0 h. On the Contrary, compared with 0 h, PYL11 relative expression was down-regulated about 3 h, 6 h and 12 h, then increased slowly, and reached the 0 h level at 24 h. Expression of PYL14 continually decreased in response to 50 µmol/L ABA. When ABA concentration doubled (Fig. 7B), the expression levels of PYL1, PYL2, PYL5, PYL8-10, PYL12 and PYL13 were all up-regulated in the four time periods, especially PYL8 at 24 h, which was 36 times higher than 0 h. PYL11 showed the same changes as 50 µmol/L ABA treatment, but decreased slightly at 3 h, 6 h and 12 h, and up-regulated at 24 h, which was 1.8 times of 0 h. Only the expression of PYL14 was slightly down-regulated at all time points.

After 200 mmol/L NaCl treatment for (Fig. 7C) 12 h, the relative expression levels of PYL5, PYL8 and PYL10 up-regulate and were 9, 24 and 10 times higher than that of 0 h respectively. Compared with 0 h, except PYL4, PYL6 and PYL11 were down-regulated at 3 h and 6 h, other genes were up-regulated in varying degrees. The relative expression of PYL14 was basically unchanged. Most of the genes were up-regulated after 10% PEG (Fig. 7D), especially PYL8 at 12 h, which was 53 times higher than 0 h. It can be seen that the relative expression of PYL genes under 10% PEG treatment is higher than other treatments, such as PYL1-5. This result showed that many CsPYL genes (especially PYL8) were likely to play critical roles in the abiotic and hormonal stress signaling transduction pathways.