Genome-Wide Analysis of the PYL Gene Family and Identification of PYL Genes That Respond to Abiotic Stress in Brassica napus.

Abscisic acid (ABA) is an endogenous phytohormone that plays important roles in the regulation of plant growth, development, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein is a core regulatory component of ABA signaling networks in plants. However, no details regarding this family in Brassica napus are available. Here, 46 PYLs were identified in the B. napus genome. Based on phylogenetic analysis, BnPYR1 and BnPYL1-3 belong to subfamily I, BnPYL7-10 belong to subfamily II, and BnPYL4-6 and BnPYL11-13 belong to subfamily III. Analysis of BnPYL conserved motifs showed that every subfamily contained four common motifs. By predicting cis-elements in the promoters, we found that all BnPYL members contained hormone- and stress-related elements and that expression levels of most BnPYLs were relatively higher in seeds at the germination stage than those in other organs or at other developmental stages. Gene Ontology (GO) enrichment showed that BnPYL genes mainly participate in responses to stimuli. To identify crucial PYLs mediating the response to abiotic stress in B. napus, expression changes in 14 BnPYL genes were determined by quantitative real-time RT-PCR after drought, heat, and salinity treatments, and identified BnPYR1-3, BnPYL1-2, and BnPYL7-2 in respond to abiotic stresses. The findings of this study lay a foundation for further investigations of PYL genes in B. napus.

1. Introduction

Abscisic acid (ABA) is an important plant hormone that plays roles not only in plant growth and development processes, such as cell division and elongation, stomatal movement, seed dormancy, embryo development, and old leaf abscission [1,2,3,4], but also in response to biotic and abiotic stresses [5]. ABA is sensed by the ABA receptor PYR/PYL (pyrabactin resistance 1-like) family in the ABA core signal transduction pathway [6,7,8]. When bound by ABA, PYR/PYL inhibits the enzymatic activity of protein phosphatase 2C (PP2C), leading to the release of the serine/threonine-protein kinase SRK2 (SnRK2) [6]. SnRK2 kinases are activated via activation loop autophosphorylation [9], and activated SnRK2 kinases subsequently phosphorylate transcription factors, such as the ABA-responsive element binding factor (ABF), which is thought to be necessary to activate ABFs [10,11]. These activated ABFs enter the nucleus to up-regulate the expression of downstream ABA-induced stress-associated genes.

Plants increase intracellular ABA content via ABA biosynthesis when subjected to abiotic stresses, such as drought, high and low temperatures, salinity, and heavy metals. Large quantities of synthetic ABA bind to PYLs, which perform ABA signal transduction and respond to stress [12]. The initial step and motivation for ABA signal transduction is ABA binding to PYLs; therefore, PYLs play important roles in this signal transduction pathway. Fourteen PYLs with highly conserved amino acid sequences have been identified in Arabidopsis thaliana and named PYR1 and PYL1-13 [6,8,13]. Furthermore, orthologous genes in other crops have been reported, including six PYLs in sweet orange [14], eight PYLs in grape [15], 21 PYL homologs in soybean [16], 12 PYLs in rice [17], 14 PYLs in tomato [18], 14 PYLs in rubber tree [19], 24 PYLs in Brassica rapa [20], and 27 PYLs in cotton [21]. These genes have been categorized into three subfamilies, and the functions of some PYL genes in plants have been characterized successfully. Overexpression of AtPYL4 in A. thaliana enhances its drought tolerance [22]. The drought and salt stress tolerance of Oryza sativa was enhanced by overexpressing OsPYL5 [23]. PYL9 promotes drought resistance, and PYL8, together with PYL9, plays a vital role in regulating lateral root growth in A. thaliana [24,25]. These results all suggest that PYL genes play a role in enhancing tolerances under abiotic stress. The identification and characterization of PYLs in these plants thus plays a very important role in understanding their function and the ABA signal transduction pathway. However, little information is available about PYLs in Brassica napus.

B. napus L. (AACC, 2n = 38), which belongs to Brassicaceae, is an allotetraploid species that are formed by an interspecific natural cross between B. rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18) and subsequent chromosome doubling approximately 7500 years ago [26]. Rapeseed is the third largest source of vegetable oil globally, after palm and soybean (http://faostat3.fao.org). B. napus provides not only high-quality oil with low levels of saturated fatty acids and cholesterol and high microelement content, but also meals for animal feed and a source of biodiesel. B. napus plants often suffer from various biotic and abiotic stresses due to environmental changes, affecting oilseed yield. Identifying PYLs in B. napus not only lays a foundation for understanding ABA signaling, but also provides information for defending against stresses.

In this study, we identified PYL genes in B. napus by protein basic local alignment search tool (BLASTP) searches of the recently completed B. napus genome [26] using the 14 PYL protein sequences from A. thaliana as queries. We analyzed phylogenetic trees, exon-intron structures, conserved protein motifs, promoter elements, and gene expression profiles in various tissues and organs, as well as Gene Ontology (GO) and micro RNA (miRNA) targeting of the BnPYL genes to further characterize BnPYLs. In addition, we analyzed the gene expression patterns of some BnPYLs under different abiotic stresses, including heat, drought, and salinity treatments. Our study provides insights into the PYL gene family in B. napus.

2. Materials and Methods

2.1. Plant Materials and Stress Treatments

Healthy B. napus ZS11 seeds were germinated on petri dishes soaked in water for 48 h and sown in 10 cm plastic pots. Seedlings were grown to the four-leaf-stage in a chamber room (16 h day/8 h dark at temperature 25 °C) and then treated with various stresses. The seedlings were irrigated with 20% polyethylene glycol-6000 (PEG-6000) or 200 mM NaCl for drought and salinity abiotic stress, respectively. Seedlings were subjected to 40 °C for high-temperature stress, and leaf samples were collected at 3, 6, and 12 h. Young leaves were collected at 3, 6, 12, 24, 48, and 72 h after drought treatment. Salinity-treated leaves were collected at 3, 6, 12, 24, and 48 h. The collected leaves were immediately frozen in liquid nitrogen and stored in a −80 °C freezer for RNA isolation.

2.2. Genome-Wide Identification and Chromosomal Location of PYL Gene Family in B. napus

To better understand the BnPYL gene family, we used 14 PYL protein sequences from the A. thaliana genome (http://www.arabidopsis.org/) as queries to identify PYL genes in B. napus, B. rapa and B. oleracea via protein basic local alignment search tool (BLASTP) [27]. The top E-value was less than 1 × 10−20. Some redundant genes were removed manually because of the complexity of the allotetraploid B. napus genome. The related gene sequences and positions were identified in BRAD (http://brassicadb.org/) and the B. napus Genome Browser (http://www.genoscope.cns.fr/brassicanapus/). The number of amino acids in a sequence and its isoelectric point (pI) and molecular weight (MW) were searched at the ExPASy website (http://web.expasy.org/). The chromosomal distributions of 46 BnPYLs were drawn using MapChart software based on their chromosomal position [28].

2.3. Phylogenetic Tree Analysis of PYL Gene Family in B. napus, B. rapa, B. oleracea and A. thaliana

To understand the evolutionary relationships of the PYL gene family, we used B. napus, B. rapa, B. oleracea, and A. thaliana protein sequences to build a phylogenetic tree. The protein sequences were multiple-aligned using MEGA 5.2 software [29]. The phylogenetic tree was built based on the neighbor-joining (NJ) method with 1000 bootstrap replicates. We then uploaded the tree diagram file (*.nwk) from MEGA to the iTOL website (http://itol.embl.de/) to better visualize the phylogenetic tree.

2.4. Analysis of Gene Exon-Intron Structures and Protein Conserved Motifs

Gene exon-intron structures were analyzed using the Gene Structure Display Server (GSDS2.0) [30] by comparing the codon sequences and genomic sequences of the 46 BnPYL members. Related gene sequences were searched on the B. napus Genome Browser (http://www.genoscope.cns.fr/brassicanapus/). Motifs were identified in Multiple EM for Motif Elicitation version 4.11.4 (MEME) [31] by analyzing 46 full-length BnPYL protein sequences. A limit of twenty motifs was set, and any number of repetitions was expected as motif sites were distributed throughout the sequences.

2.5. Analyzing cis-Elements in the BnPYL Promoters

We analyzed the cis-elements of BnPYL promoters to further understand the BnPYL gene family. We examined the sequences within 1500 base pairs (bp) upstream of initiation codons (ATG) for promoter analysis and were searched for these sequences in the B. napus Genome Browser. The cis-elements in promoters were subsequently searched using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

2.6. Prediction of miRNAs Targeting BnPYL Genes

In this study, all of the genome sequences of BnPYL family genes were submitted as candidate genes for predicting potential miRNAs by searching against the available B. napus reference of miRNA sequences using psRNATarget Server with default parameters [32]. Cytoscape software [33] was used to visualize the interactions between the predicted miRNAs and the corresponding target BnPYL genes.

2.7. Analysis of Gene Expression Profiles and Gene Ontology Enrichment

To further characterize the different temporal and spatial gene expression patterns of the BnPYL gene family, we analyzed RNA sequencing (RNA-seq) data. Transcriptome sequencing datasets were deposited in the BioProject ID PRJNA358784, which was used to perform RNA-seq of different B. napus cultivar ZS11 tissues. We analyzed the total RNA-seq data of the roots, stems, leaves, flowers, seeds, and siliques at the germination, seedling, bud, initial flowering, and full-bloom stages. We quantified these gene expression levels on the basis of their fragments per kilobase of exon per million reads mapped (FPKM) values using Cufflinks with default parameters [34], and represented these results using HemI 1.0 software [35]. To further understand the functions of these genes, we used BLAST software (https://blast.ncbi.nlm.nih.gov/) to align the BnPYL sequences with entries in the NCBI nonredundant (NR) protein [36], Swiss-Prot [1], GO [37], clusters of orthologous groups (COG) [38], eukaryotic orthologous groups (KOG) [39], Protein family (Pfam) [40], and Kyoto encyclopedia of genes and genomes (KEGG) databases [41], and conducted GO enrichment analysis of those BnPYLs that were annotated. GO enrichment was performed using the BinGO program of Cytoscape_3.4.0 software [33] with an FDR < 0.01.

2.8. RNA Extraction and Real-Time RT-PCR

RNA was extracted from drought-, heat- and salinity-treated samples using an EZ-10 DNAaway RNA Mini-prep Kit (Sangon Biotech, Shanghai, China), according to the manufacturer’s instructions. RNA concentrations were measured by a NanoDrop 2000 (Thermo Fisher Scientific, Worcester, MA, USA), and RNA integrity was evaluated by electrophoresis. One microgram of RNA template from each sample was used to synthesize the first-strand of complementary DNA (cDNA) using an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and the cDNA solution was then diluted 20 times with distilled deionized water. Each reaction had a final volume of 20 µL and contained 2 µL of 20-fold-diluted cDNA solution, 10 µL of SYBR® Green Supermix (Bio-Rad), 0.4 µL of 10 mM forward and reverse primers, and 7.2 µL of distilled deionized water. We performed qRT-PCR on a CFX96 Real-time System (Bio-Rad), according to the manufacturer’s instructions. The qRT-PCR program was as follows: 98 °C for 30 s, then 40 cycles of 98 °C for 15 s, 60 °C for 30 s, and an increase from 65 °C to 95 °C with an increment of 0.5 °C every 0.05 s. Three technical replications were performed per sample. We calculated the relative gene expression levels based on the 2−ΔΔCt method using Actin7 of B. napus as an internal control for normalizing gene expression levels. RT-PCR primers were designed on Primer Premier Software (version 5.0). Given the highly homologous candidates in B. napus, all of the primer sequences avoided false priming and are listed in Table S1. All of the qRT-PCR results were displayed using HemI 1.0 software [35].

3. Results

3.1. Characterization of BnPYL Gene Family

In this study, we identified 46 PYL genes in the B. napus genome through BLASTP by using 14 AtPYL protein sequences as references (Table 1). Every member of the 14 AtPYLs was homologous to one to six sequences in B. napus genome. For example, AtPYL4, AtPYL6 and AtPYL8 had six homologs in B. napus, but AtPYL11 and AtPYL12 had only one homolog. We found that 46 BnPYL members were all derived from a progenitor by comparing the composition of these PYL genes in B. napus and their relatives in its two ancestors B. rapa and B. oleracea. The B. rapa genome contains 22 BnPYLs and the B. oleracea genome contains 24 BnPYLs. Based on the physical positions of the 46 BnPYL genes, 38 BnPYLs were accurately mapped onto the 19 B. napus chromosomes, whereas the remaining BnPYLs were located on the unmapped scaffolds in the Ann_random and Cnn_ random genomes (Figure 1). BnPYLs are distributed on all B. napus chromosomes, except A07, A08, and C06, and were densely distributed on A03 and C03, containing five and six members, respectively (Figure 1). Table 1 shows that the gene lengths range from 489 (BnPYL11) to 2229 (BnPYL8-3), with one to four exons in each sequence. The transcripts, except for introns and noncoding regions (UTR), consist of coding DNA sequences (CDS) with sizes varying from 489 (BnPYL11) to 648 bp (BnPYL1-1). The lengths of the corresponding BnPYL protein sequences range from 162 (BnPYL11) to 215 (BnPYL1-1) amino acids. The average MW was 21.64 kDa. The pI values of these proteins range from 5.12 (BnPYL13-1) to 8.91 (BnPYL3-3), and 89.13% of these proteins are acidic (pI < 7).

Table 1

PYL gene family information in Brassica napus.

Gene ID	Gene Name	Position (bp)	Gene Length (bp)	CDS Length (bp)	Exon	Peptide Residues	MW (kDa)	pI
BnaCnng65400D	BnPYR1-1	65121492-65122292	801	576	1	191	21.69	6.39
BnaC07g34880D	BnPYR1-2	37423726-37424722	997	576	1	191	21.40	5.98
BnaAnng35310D	BnPYR1-3	40055219-40055779	561	561	1	186	21.11	6.39
BnaA03g43410D	BnPYR1-4	21808356-21809284	929	576	1	191	21.37	5.98
BnaC07g19450D	BnPYL1-1	26187392-26188931	1540	648	1	215	24.38	5.29
BnaA06g40360D	BnPYL1-2	200261-2003467	854	633	1	210	23.91	5.20
BnaA09g40690D	BnPYL2-1	28565054-28565746	693	567	1	188	20.83	5.49
BnaC08g33170D	BnPYL2-2	31754064-31754755	692	576	1	188	21.03	5.86
BnaC02g21700D	BnPYL3-1	18634088-18634705	618	618	1	205	22.91	8.87
BnaC03g23260D	BnPYL3-2	12925626-12926243	618	618	1	205	22.91	8.88
BnaA02g16230D	BnPYL3-3	9682751-9683368	618	618	1	205	22.88	8.91
BnaAnng13200D	BnPYL4-1	14172422-14173048	627	627	1	208	22.57	7.08
BnaA04g21960D	BnPYL4-2	16651226-16652092	867	615	1	204	21.99	6.22
BnaA03g17720D	BnPYL4-3	8353447-8354313	867	624	1	207	22.24	6.43
BnaC04g07010D	BnPYL4-4	5159788-5160411	624	624	1	207	22.42	7.08
BnaC03g21240D	BnPYL4-5	11473885-11474748	864	624	1	207	22.29	6.43
BnaC04g56560D	BnPYL4-6	4191879-4192790	912	615	1	204	21.94	6.04
BnaA10g24990D	BnPYL5-1	16198838-16199452	615	615	1	204	22.78	6.02
BnaC09g49910D	BnPYL5-2	48035079-48036202	1124	615	1	204	22.77	5.82
BnaAnng40650D	BnPYL5-3	46553930-46554584	655	612	1	203	22.71	5.80
BnaC03g02130D	BnPYL5-4	994403-995014	612	612	1	203	22.68	5.91
BnaAnng01330D	BnPYL5-5	811157-811750	594	594	1	197	22.02	5.91
BnaA05g05420D	BnPYL6-1	2798876-2799514	639	639	1	212	23.48	6.52
BnaA03g19030D	BnPYL6-2	9003640-9004337	698	639	1	212	23.52	6.56
BnaA04g29300D	BnPYL6-3	1303160-1304112	953	618	1	205	22.77	6.09
BnaC03g22610D	BnPYL6-4	12499410-12500051	642	642	1	213	23.71	6.38
BnaC04g47050D	BnPYL6-5	46155948-46156559	612	612	1	203	22.50	6.10
BnaC04g04830D	BnPYL6-6	3526371-3527009	639	639	1	212	23.50	6.66
BnaC03g31730D	BnPYL7-1	19523342-19524503	1162	582	1	193	21.69	6.05
BnaA03g26790D	BnPYL7-2	13185542-13186722	1181	582	1	193	21.80	6.24
BnaC02g14540D	BnPYL8-1	10060041-10061474	1434	567	3	188	21.32	6.71
BnaA03g12450D	BnPYL8-2	5667935-5670048	2114	552	4	183	20.67	6.24
BnaC03g15210D	BnPYL8-3	7539236-7541464	2229	555	3	184	20.80	6.24
BnaA02g10420D	BnPYL8-4	5349136-5350394	1259	567	3	188	21.26	6.24
BnaA10g06520D	BnPYL8-5	4967767-4969159	1393	555	3	184	21.03	6.07
BnaCnng37890D	BnPYL8-6	36425463-36426990	1528	555	3	184	20.93	6.07
BnaC05g00620D	BnPYL9-1	331808-333031	1224	564	3	187	21.03	5.98
BnaC05g17260D	BnPYL9-2	10921616-10922842	1227	561	3	186	21.02	5.98
BnaA10g00540D	BnPYL9-3	270030-271246	1217	567	3	188	21.20	6.06
BnaA01g16740D	BnPYL10-1	8730925-8731696	772	558	3	185	20.78	5.61
BnaCnng68710D	BnPYL10-2	68363157-68363922	766	558	3	185	20.73	5.71
BnaA06g40220D	BnPYL11	1928871-1929359	489	489	1	162	17.82	5.40
BnaCnng60010D	BnPYL12	59845750-59846238	489	489	1	162	17.83	5.40
BnaC01g11020D	BnPYL13-1	6873982-6874482	501	501	1	166	18.41	5.12
BnaA01g09460D	BnPYL13-2	4636999-4637499	501	501	1	166	18.40	5.26
BnaC07g48850D	BnPYL13-3	1477983-1478483	501	501	1	166	18.41	5.26

MW: molecular weight; pI: isoelectric point.

Figure 1

BnPYL distributions on B. napus chromosomes. The chromosome name is at the top of each bar. Ann_random: unmapped A chromosomes of the B. napus genome; Cnn_random: unmapped C chromosomes of the B. napus genome; the scale of the chromosome is in millions of bases (Mb).

3.2. Analysis of Phylogenetic Relationships and Gene Structures of BnPYLs

To study the evolutionary relationships between BnPYLs and PYLs from A. thaliana, B. rapa and B. oleracea, the 46 BnPYLs with 14 AtPYL, 22 BoPYL, and 20 BrPYL were clustered into three groups, designated Group I, Group II, and Group III, on an unrooted phylogenetic tree, with at least 67% bootstrap support for Group III. PYR1 and PYL1-3 belong to Group I, PYL7-10 belong to Group II, and PYL4-6 and PYL11-13 belong to Group III (Figure 2). Overall, Group I contained 25 members, Group II contained 30 PYLs, and Group III comprised 47 PYLs. Specifically, 11, 13, and 22 BnPYLs were found to be distributed into Groups I, II, and III, respectively. PYLs grouping into the same subfamilies may have similar functions. To understand the PYL gene structures, we analyzed the BnPYL gene exon-introns using the GSDS website. We presented these structural features based on evolutionary tree relationships. In Figure 3, Groups I and III do not possess introns, and all of the Group II members, except BnPYL8-2, have two introns each. BnPYL8-2 contains three introns (Figure 3). These results indicated that members within a single subfamily had highly similar gene structures, which is consistent with their phylogenetic relationships.

Figure 2

Phylogenetic tree analysis of PYLs in Arabidopsis thaliana, B. napus, Brassica rapa and Brassica oleracea. In total, 14 AtPYLs from A. thaliana, 24 BrPYLs from B. rapa, 23 BoPYLs from B. oleracea and 46 BnPYLs from B. napus were included. These 107 sequences were used to construct a neighbor-joining (NJ) tree. The tree was divided into three groups, represented by different colors.

Figure 3

The exon-intron structure of BnPYLs according to their phylogenetic relationships. Light yellow BnPYLs represent Group III; BnPYLs in light purple belong to Group II; and BnPYLs in light blue are Group I. The lengths and positions of introns and exons are shown on the figure. The green boxes and gray lines denote exons and introns, respectively. CDS: coding sequences; bp: base pairs.

3.3. Analysis of BnPYL Conserved Motifs

We analyzed full-length protein sequences of 46 BnPYLs using MEME software to identify their conserved motifs. Twenty conserved motifs were recognized, and the length of the motifs range from 8 to 41 amino acids. Every BnPYL member contains from four to eight conserved motifs (Figure 4). Motifs 1, 2, and 3 are present in all 46 BnPYL proteins, and three motifs contained a START-like domain. All the proteins except BnPYL8-2 show motif 4. Furthermore, all the members of Group II contain motif 8. We found that every subfamily possesses four identical motifs, suggesting that PYL proteins have highly conserved amino acid residues and members of the same group may have similar functions. In addition, we compared the three motifs that are common to all BnPYL proteins with sequences from another study and found that those amino acids marked with asterisks in Figure S1 are similar to the sequences in the previous study [20], suggesting that these residues may be play a role in receptor activation.

Figure 4

The conserved motifs of the BnPYL proteins presented according to their phylogenetic relationships. These motifs were identified using Multiple EM for Motif Elicitation (MEME), and boxes of different colors represent different motifs.

3.4. Cis-Elements in BnPYL Promoters

To better understand the transcriptional regulation and potential function of the BnPYL genes, we isolated sequences within 1500 bp upstream of the initiation codons of BnPYLs and identified cis-elements within these promoter sequences using the PlantCARE database. We analyzed ten hormone-related and five stress-related elements (Table S2). The upstream regions of all BnPYL members contain at least two hormone-related elements, such as abscisic acid-responsive (ABRE), auxin-responsive (AUXRR-core, TGA-element), MeJA-responsive (CGTCA-motif, TGACG-motif), ethylene-responsive (ERE), gibberellin-responsive (GARE, P-box, TATC-box), and salicylic acid-responsive elements (TCA-element). In addition, the 46 BnPYL promoters contain one or more stress-related elements, such as fungal elicitor-responsive (Box-W1/W3), heat stress-responsive (HSE), and low-temperature-responsive (LTR) elements and a MYB-binding site that is involved in drought-inducibility (MBS), defense, and stress responsiveness (TC-rich repeats). The four most abundant hormone-related elements in the 46 BnPYLs are ABRE, CGTCA-motif, TGACG-motif, and TCA-element, and the three most abundant stress-related elements are HSE, MBS, and TC-rich repeats. The MBS element was found in 38 of 46 BnPYLs, suggesting that BnPYLs may play an important role in regulating drought stress. BnPYL8-3 contains ten MeJA-related elements, suggesting that BnPYL8-3 may respond to MeJA exposure. Similarly, BnPYL8-5 contains seven MBS elements and may be related to drought tolerance. The diversities of the hormone- and stress-related cis-elements in the BnPYL promoters suggested that expression may differ in response to hormones and stresses.

3.5. Comprehensive Analysis of microRNA Targeting BnPYL Genes

In recent years, a considerable number of studies have shown that miRNAs mainly respond to stress by regulating the expression of genes associated with stress in plants. To understand the underlying regulatory mechanism of miRNAs involved in the regulation of BnPYLs, we identified 26 putative miRNAs targeting 11 BnPYL genes to construct a relationship network using Cytoscape software (Figure 5). We analyzed the connection distribution of the regulation network and found BnPYR1-2 and BnPYR1-4 are the most targeted BnPYL genes for successful targeting by B. napus miRNAs. Ten members of the miRNA169 family and four members of the miRNA172 family target BnPYR1-2, and 10 members of the miRNA169 family target BnPYR1-4. Notably, miR169 plays important roles in A. thaliana by targeting genes related to drought stress [42]. In addition, BnPYL2-1, BnPYL2-2, BnPYL4-3, BnPYL4-5, BnPYL6-3, BnPYL6-5, BnPYL8-1, BnPYL9-3, and BnPYL10-2 were regulated by different miRNAs. Furthermore, miR167d was identified as an miRNA targeting four BnPYL genes (BnPYL4-3, BnPYL4-5, BnPYL6-3, and BnPYL6-5), the most targeted BnPYL genes in our study.

Figure 5

A schematic representation of the regulatory network relationships between the putative miRNAs and their targeted BnPYL genes.

3.6. Analysis of BnPYL Expression Levels in Tissues

To characterize the expression of the BnPYL gene family, we analyzed 50 different tissues and organs of B. napus at different development stages based on RNA-seq datasets from B. napus ZS11 (BioProject ID PRJNA358784); the reliability of the datasets was verified by Zhou et al. using qRT-PCR [34]. The expression levels of most PYL members differed in the different tissues and organs, suggesting that different functions were required in different tissues. Notably, the expression levels of most PYL genes in seeds (roots, hypocotyl, cotyledon and germinated seed) at the germination stage were higher than those of other organs and of plants at other developmental stages (Figure 6 and Table S3). Thirteen BnPYL genes (PYR1-2, PYR1-4, PYL1, PYL4-2, PYL4-6, PYL5-2, PYL5-4, PYL6-3, PYL6-5, and PYL9) were highly expressed in nearly all tissues, suggesting that these genes play an important role in regulating B. napus biology process. By contrast, the expression abundances of 14 PYLs (PYL2, PYL3, PYL4-1, PYL5-5, PYL10, PYL11, PYL12, and PYL13) in all tissues were very low, with nearly no expression in B. napus. The expression levels of the same gene in the same tissues or organs differed in different growth stages, suggesting that some genes are expressed at specific times.

Figure 6

Expression levels of BnPYL genes in different tissues and at different stages of B. napus. Ro, root; St, stem; Le, leaf; Hy, hypocotyl; Ao, anthocaulus; Cal, calyx; Cap, capillament; Pe, petal; Sta, stamen; Pi, pistil; SP, silique; Se, seed; SC, seed coat; Em, embryo; Co., cotyledon; GS, germinate seed. s, seedling stage; b, bud stage; i, initial flowering stage; and, f, full-bloom stage. The 24, 48, and 72 h labels indicate the time that had passed after seed germination. The 3, 19, 21, 30, 40, and 46 d labels indicate the number of days that had passed after the flowering stage. The bar on the lower right corner represents fragments per kilobase of exon per million reads mapped (FPKM) values, and different colors represent different expression levels.

3.7. Gene Ontology Enrichment

To further understand the functions of the BnPYLs, we performed GO annotation and GO enrichment analyses. The GO terms included three categories, biological process (BP), molecular function (MF), and cellular component (CC). GO enrichment confirmed that these 46 BnPYLs are enriched in the cell (GO:0005623), cell part (GO:0044464) and organelle (GO:0043226) terms of the CC category. MF is enriched in binding (GO:0005488). Biological regulation (GO:0065007) and response to stimulus (GO:0050896) were the most abundant functions in the BP category (Table S4). The GO enrichment suggested that BnPYLs play important roles in responding to stress, consistent with the findings of a previous study [5].

3.8. The Expression Patterns of PYLs in B. napus under Abiotic Stress

To further explore BnPYL gene expression patterns under abiotic stresses and identify genes important for improving tolerance to abiotic stresses, B. napus seedlings were subjected to abiotic stresses such as drought, salinity, and heat. A total of 14 BnPYLs were selected to perform quantitative real-time RT-PCR at different time points after various abiotic treatments, and the expression levels of these genes are listed in Table S5. The expression patterns of selected 14 BnPYL genes showed transcriptional changed under drought, heat, and salinity stresses, and this suggested that the response of BnPYLs to multiple stresses is a dynamic process (Figure 7). For drought treatment, three genes (PYR1-3, PYL1-2 and PYL7-2) of the 14 selected genes had similar expression patterns; specifically, they tended to be up-regulated at all of the time points. The expression levels of PYL3-1, PYL6-1, PYL8-5, and PYL8-6 increased at one or three time points. The seven up-regulated genes (PYR1-3, PYL1-2, PYL3-1, PYL6-1, PYL7-2, PYL8-5, and PYL8-6) were highly induced in response to drought treatment at 12 h than at other time points. The change of PYR1-4 expression levels is not significantly at all time points under drought stress. By contrast, the expression levels of other six BnPYL genes (PYL2-2, PYL4-2, PYL4-6, PYL5-4, PYL9-1, PYL9-2) were generally down-regulated under drought treatment. For the up-regulated BnPYL genes after drought treatment, they may be related to drought tolerance. Under heat stress, 14 BnPYL genes showed different expression levels (Figure 7). PYR1-3, PYL1-2, PYL3-1, PYL4-2, PYL5-4, PYL6-1, PYL7-2, and PYL 9-2 were up-regulated, and the expression levels of PYR1-3 at 6 h and PYL7-2 at three-time points considerably up-regulated under heat treatment. The other genes were down-regulated after heat treatment. For salinity stress, PYR1-3, PYL1-2, PYL3-1, PYL7-2, PYL8-5, and PYL8-6 were up-regulated at specific time points or during periods of time, while the other genes were down-regulated. The expression level of PYL3-1 was stable or down-regulated in the 24 h after treatment, but it was up-regulated at 48 h, which suggested that some PYLs may be induced under serious stress conditions. The expression levels of PYR1-3 and PYL1-2 increased during the early stages, stabilized at 12 h and increased at 24 and 48 h, which showed the complexity of the regulatory networks in responding to stresses. Taken together, we found the expression patterns of some BnPYL genes are similar under drought, salinity, and heat treatments (Figure 7). In addition, our results suggested that the expression levels of PYR1-3, PYL1-2, and PYL7-2 were induced by drought, high-temperature, and salinity stresses, suggesting that these genes might be important candidates for improving tolerance to abiotic stresses.

Figure 7

PYL expression levels under drought, salinity and heat abiotic stresses in B. napus. The bars display the relative gene expression levels, calculated based on the 2−ΔΔCt method. The expression level is equal to the mean values and transform log2 values. polyethylene glycol (PEG), drought stress; Heat, high-temperature stress; Salinity, salt stress.

4. Discussion

The phytohormone ABA is well known for its two functions: the regulation of plant growth and development and the responding to abiotic and biotic stresses. In our research program, we had a wilting mutant of B. napus through Ethyl methanesulfonate (EMS) mutagenesis. The wilting mutant accumulated a higher content of ABA than the wild type, and the expression of PYL genes were up-regulated compared with wild type. PYL as ABA receptor is the first step for the downstream ABA signaling, and an important element in the core ABA signal transduction pathway. Although PYLs have been identified in many plants, this work is the first identification of PYL genes in B. napus.

4.1. Characterization of PYL Gene Family in Brassica napus

B. napus as an allotetraploid species that experienced widespread genome duplication and merging events [26]. According to our results, the number of PYL genes in B. napus far exceeds the 14 AtPYLs in A. thaliana [8], suggesting that genome duplication may have occurred in the evolution of B. napus. Each PYL in A. thaliana was typically homologous to 2-6 genes in the B. napus genome, consistent with the finding that one A. thaliana gene corresponded to two or more homologous genes in B. napus [43]. Based on phylogenetic analysis, the 46 BnPYLs were classified into the same three groups as PYL genes from A. thaliana (Figure 2), suggesting similar evolutionary trajectories between B. napus and A. thaliana. In addition, the results indicated that the encoding BnPYL genes, which are homologous to A. thaliana genes, might play similar roles in specific biological processes. These groupings in the phylogenetic tree were supported by the exon-intron structure (Figure 3). These clusters of subfamilies were consistent with the groupings of B. rapa and tomato [18,20], but were different from the groupings of the rubber tree genes, which were divided into the groups HbPYL1-3, HbPYL4-7, and HbPYL8-14 [19]. This difference may be caused by the sequence diversity of the various species. In addition, the numbers and composition of motifs were varied in each BnPYL family. Motif 1, motif 2, and motif 3 with 41, 41, and 37 amino acid residues, respectively (Figure S1) were detected in all BnPYL protein sequences (Figure 4), indicating that BnPYLs have a highly conserved protein structures. Phylogeny analysis of BnPYL genes is sharing the similar motifs in each subfamily (Figure 4). However, BnPYL genes of the same group have similar functions, although we do not know the functions of these groups.

4.2. Expression Levels of BnPYLs in Various Tissues

PYL gene expression patterns in different tissues have been reported for many plants. In soybean, most PYLs are expressed at relatively higher levels in seeds than those in other soybean tissues [16]. In rubber tree, transcripts of PYLs are highly abundant in latex [19]. PYLs in B. rapa are expressed at a higher level in the callus than those in other tissues [20]. We analyzed the PYL gene expression patterns in the various tissues of oilseeds and found that BnPYLs show very high expression levels in seeds during germination stage (Figure 6). These results suggest that PYLs play important roles in regulating seed germination in B. napus, and that ABA signaling also participates in the regulation of seed germination in B. napus. The expression levels of 14 BnPYLs (BnPYL2, BnPYL3, BnPYL4-1, BnPYL5-5, BnPYL10, BnPYL11, BnPYL12, and BnPYL13) in all of the tissues were nearly zero in B. napus, suggesting that the functions of these genes are not required.

4.3. The Expression Patterns of BnPYLs under Abiotic Stresses

B. napus faces multiple abiotic stresses such as drought, high temperature and salinity, which seriously affect oilseed yields and seed quality. ABA has recently been reported to play crucial roles in responding to abiotic stresses, such as drought and salinity [44,45,46]. PYLs are involved in the initial step in ABA signal transduction. However, it is still unknown which PYL is the important ABA receptor in response to abiotic stress in B. napus. In our study, we selected 14 BnPYLs for qRT-PCR analysis and found that PYR1-3, PYL1-2, and PYL7-2 were induced by heat, drought, and salinity stress, respectively (Figure 7), suggesting that these PYLs have multifunctional roles under various abiotic stresses. In addition, PYL8-5 and PYL8-6 were up-regulated in B. napus under drought stress, which is consistent with the results in cotton. In cotton, GhPYL26, which is homologous to the PYL8 gene in A. thaliana, is overexpressed to enhance drought tolerance [21]. In a study by Zhao et al., AtPYL9 overexpression promoted drought resistance in A. thaliana [25], whereas the expression levels of BnPYL9-1 and BnPYL9-2 were inhibited by drought and salinity stress. We thus speculated that this behavior may be explained by a negative-feedback regulatory mechanism: when a large quantity of ABA accumulates in the leaves under stress, PYL expression may be inhibited [47,48]. By contrast, the expression patterns of 14 BnPYLs under drought and salinity stress were similar. ABA is produced rapidly in response to stress under drought and salinity conditions and then plays an important role in the regulation of the stress response [49]. These findings and our data suggest that the PYL response mechanisms under drought and salinity stress may be similar. In conclusion, the results of the stress response experiments, combined with the analysis of the stress-responsive cis-elements in BnPYL promoters, suggest that some BnPYLs respond to drought, high temperature, and salinity treatments. These PYLs may potentially be utilized for improving the tolerance of B. napus to abiotic stresses.