Skip to main content
Download PDF

Genome-wide identification and characterization of ABA receptor PYL gene family in rice

BMC Genomics volume 21, Article number: 676 (2020) Cite this article

Abstract

Background

Abscisic acid (ABA), a key phytohormone that controls plant growth and stress responses, is sensed by the pyrabactin resistance 1(PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Comprehensive information on evolution and function of PYL gene family in rice (Oryza sativa) needs further investigation. This study made detailed analysis on evolutionary relationship between PYL family members, collinearity, synteny, gene structure, protein motifs, cis-regulatory elements (CREs), SNP variations, miRNAs targeting PYLs and expression profiles in different tissues and stress responses.

Results

Based on sequence homology with Arabidopsis PYL proteins, we identified a total of 13 PYLs in rice (BOP clade) and maize (PACCMAD clade), while other members of BOP (wheat – each diploid genome, barley and Brachypodium) and PACCMAD (sorghum and foxtail millet) have 8-9 PYLs. The phylogenetic analysis divided PYLs into three subfamilies that are structurally and functionally conserved across species. Gene structure and motif analysis of OsPYLs revealed that members of each subfamily have similar gene and motif structure. Segmental duplication appears be the driving force for the expansion of PYLs, and the majority of the PYLs underwent evolution under purifying selection in rice. 32 unique potential miRNAs that might target PYLs were identified in rice. Thus, the predicted regulation of PYLs through miRNAs in rice is more elaborate as compared with B. napus. Further, the miRNAs identified to in this study were also regulated by stresses, which adds additional layer of regulation of PYLs. The frequency of SAPs identified was higher in indica cultivars and were predominantly located in START domain that participate in ABA binding. The promoters of most of the OsPYLs have cis-regulatory elements involved in imparting abiotic stress responsive expression. In silico and q-RT-PCR expression analyses of PYL genes revealed multifaceted role of ABARs in shaping plant development as well as abiotic stress responses.

Conclusion

The predicted miRNA mediated regulation of OsPYLs and stress regulated expression of all OsPYLs, at least, under one stress, lays foundation for further validation and fine tuning ABA receptors for stress tolerance without yield penalty in rice.

Background

Abscisic acid (ABA) plays a pivotal role in plant growth and development including cell elongation and division, embryo maturation, desiccation tolerance of seeds, seed dormancy, germination, leaf senescence, induction of root growth and fruit ripening. In addition ABA regulates stomatal aperture [1,2,3,4,5,6] and is the primary hormone imparting cellular tolerance to various biotic and abiotic stresses in plants [7,8,9,10].

Over the past one decade mammoth advancements have been made in unravelling the mechanism of ABA signalling including the discovery of ABA receptors [11]. The PYLs are currently the largest plant hormone receptor family known [12]. ABA is perceived by soluble cytosolic pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/ Regulatory component of ABA receptor (RCAR) protein family in Arabidopsis [13,14,15]. Binding of ABA to PYL leads to conformational change in PYL enabling it to bind to clade A type 2C protein phosphatases (PP2Cs). Binding of PYL to PP2C releases class III sucrose non-fermenting 1-related protein kinase 2 s (SnRK2s) from inhibition by PP2Cs [4, 11,12,13, 15,16,17,18,19,20,21,22]. Activated SnRK2s phosphorylate downstream targets like Abscisic acid responsive element (ABREs)/Abscisic acid binding factor (ABF)/ABI5 clade of bZIP transcription factors and other regulatory proteins promoting ABA induced physiological responses [16,17,18,19]. Thus, trinity of PYLs, PP2Cs and SnRKs constitute the core ABA signalling modules which are highly conserved in land plants which need abiotic stress tolerance for survival [15,16,17,18,19,20,21,22,23,24,25,26].

Voluminous efforts have been made in characterization of PYL receptors from model plant Arabidospsis which encodes for 14 PYL members that are highly conserved in amino acid sequence as well as in functional domain structure [13, 14, 27]. AtPYR1, AtPYL1 and AtPYL2 are dimeric, while AtPYL4 to AtPYL10 are monomeric in apo-receptor state. AtPYL3 exists in both monomeric and dimeric state. PYL receptors negatively regulate PP2Cs in an ABA independent manner [20]. Based on sequence similarity, ABA receptors of Arabidopsis have been broadly classified into 3 subfamilies [13]. ABA receptors belonging to subfamily I and II are monomeric, while subfamily III are dimeric in nature. The overall PYL structure exhibits the helix-grip fold, a hallmark of START (star-related lipid transfer) domain/Bet v 1-fold proteins, which is characterised by the presence of a central β-sheet surrounded by N- and C-termini α-helices, with a long C terminal α-helix packing tightly against the β-sheet. The helix-grip fold creates a large cavity constituting the ABA binding pocket [20, 23].

Extensive studies with PYLs in Arabidopsis have shown that PYL family ABA receptors play a diverse role in plant development and combating abiotic stresses. AtPYR1, AtPYL1, AtPYL2, AtPYL4, AtPYL5, AtPYL8 and AtPYL9 have been shown to promote ABA induced seed germination, stomatal closure and root growth; AtPYL6 and AtPYL13 have been shown to inhibit seed germination [13, 28,29,30,31]. Apart from playing key role in growth and development, AtPYL5 and AtPYL9 were found to provide drought tolerance [15, 32].

Since the discovery of PYL family of ABA receptors, a lot of efforts have been made to unravel the function of PYL members in diverse plant species and agriculturally important crops, including Arabidopsis [15, 22, 28, 32,33,34,35,36,37], Artemisia annua [38], Vitis vinifera [39, 40], Oryza sativa [41,42,43,44,45,46,47], Triticum aestivum [48, 49], Zea mays [50, 51], Solanum lycopersicum [52], Glycine max [53], Populus [54], Hevea brasiliensis [55], strawberry [56], Gossypium hirsutum [57,58,59], Brassica rapa [60] and Brachypodium distachyon [61, 62]. As compared with dicotyledonous model plant Arabidopsis, signalling modules in the monocot rice are similar in type and number which proves the conserved nature of functional ABA signalling pathway across plant species [49, 63,64,65].

Abiotic stresses such as cold, drought and salinity have detrimental effect on the agricultural crops leading to yield losses worldwide [66]. Despite the advancements achieved towards the comprehension of the role of PYL gene family in Arabidopsis, functional diversity and redundancy of PYLs in development and stress responsive processes in agronomically important crop like rice is relatively less investigated. In rice OsPYL2, OsPYL8, OsPYL9, OsPYL10 and OsPYL11 receptors have been functionally characterized by overexpressing the PYL genes while CISPR/Cas9 knockout mutants of PYL genes in rice has been found to moderate abiotic stress tolerance and yield [41,42,43,44,45,46,47]. Most of these works were focused in japonica rice while very less information is available in indica rice. Most of the mentioned approaches involved characterization using either overexpressing or knocking down individual OsPYL gene. Moreover previous expression studies of PYL genes were confined to a particular stage and stress. A detailed genome wide analysis of ABARs in terms of evolution, promoter analysis, miRNA targets and functional characterization has not been reported in rice. Functional validation of ABA receptor is pivotal for plant genetic engineering towards improving important agricultural traits such as plant biomass, yield and tolerance to abiotic stresses.

In the present study, genome wide identification and characterization of PYL gene family in Oryza sativa spp. indica was carried out using comparative genomic tools and experimental verification. A total of 13 OsPYL genes were identified from rice genome. Further we discerned PYL gene family in other agriculturally imperative crops and phylogenetic relationship amongst them. Genomic organization, gene structure, motif composition, subcellular localization and miRNA targets and expression analysis were characterized using in silico approaches. Collinearity and syntenic relationship of PYL gene across different taxas was studied. Non synonymous SNPs were identified across popular rice accessions to study polymorphism amongst them. We also carried out a detailed investigation of cis-regulatory elements in promoter region of ABA receptors in relation to their role in stress responsiveness and development. A comprehensive differential gene expression profiling of OsPYL gene family in spatiotemporal manner was carried out under different stresses (drought, ABA, low temperature, salinity, high temperature) and tissues using in silico data and quantitative PCR analysis. Our results provide a foothold in understating functions to further illuminate OsPYL genes under different stresses and development and identification of targets for improving abiotic stress tolerance in rice.

Results

Genome-wide identification and phylogenetic analyses of the OsPYL gene family in rice

To identify all the OsPYL gene members in rice, Hidden Markov model and BLASTp (e-value <=1e-10) searches were carried out to search rice genome annotation project (RGAP) (http://rice.plantbiology.msu.edu/) using 14 Arabidopsis PYL amino acid sequence as queries [67, 68]. A total of 13 OsPYL genes were identified in the genome of rice. Nomenclature of the identified OsPYL genes was done in accordance with previous study of OsPYLs [45]. Among these, two (OsPYL7 and OsPYL12) are thought to be non-functional ABA receptors as they have large deletion in the N and C terminal of the gene, respectively [26]. The identified rice OsPYL genes encode protein with size ranging from 125 (OsPYL12) to 229 (OsPYL6) amino acid residues. The other characteristics of the OsPYL genes, including gene length, open reading frame (ORF) length, the isoelectric point (pI), molecular weight (MW), and exons, are presented in (Table 1, Additional files 1, 2).

Table 1 Basic information of OsPYL family genes and their proteins in Oryza sativa spp. indica
Full size table

To understand the phylogenetic relationship of the OsPYL proteins, maximum likelihood method was used and tree was constructed using MEGA X [69, 70]. The OsPYL proteins could be grouped into three major subfamilies viz., sub-family I, II and III based on sequence similarity (Fig. 1a). Among the 13 OsPYL proteins, OsPYL7 to OsPYL13 (seven PYLs) belong to subfamily I; OsPYL4 to OsPYL6 belong to subfamily II (three PYLs), while OsPYL1 to OsPYL3 belong to subfamily III (three PYLs).

Fig. 1
figure 1

Phylogenetic relationship, gene architecture and conserved motifs of PYL genes. a Phylogenetic relationship of PYLs from in Arabidopsis and rice. Tree was constructed by the Maximum likelihood method. The blue, green, and red boxes depict the subfamily I, II and III, respectively. b Exon/intron architectures of PYLs. Grey colour boxes indicate exons and lines represent introns. The lengths of the exons and introns for each PYL gene can be calculated following the scale at the bottom. c Distributions of conserved motifs in PYL proteins. Motifs are indicated by 10 different colour boxes. d Legend depicting the protein sequence of the corresponding motifs

Full size image

Gene structure of OsPYL genes

Gene Structure Display Server (GSDS v2.0) [71] analysis of intron/exon structure of AtPYLs and OsPYLs (Fig. 1b) showed that the AtPYLs and OsPYLs can be distinctly grouped into intronless clade and a clade having introns. No introns were detected in the PYL genes of subfamilies II and III, whereas all of the members in subfamily I consists of two introns except OsPYL7 and OsPYL12. Different OsPYL genes were grouped along with their homologous AtPYL genes in each subfamily depicting similar exon-intron structures, which affirms their close evolutionary relationships and the classification of subfamilies.

Conserved motifs of OsPYLs

Among 13 OsPYLs, motif 1 harbouring the trademark Gate–Latch domain was conserved across all PYLs (Fig. 1c and Fig. 1d), while motif 2 and 3 were found to be conserved among all receptors except OsPYL7 and OsPYL12 (Table 1). OsPYLs have two (OsPYL12) to six (OsPYL2, 3, 8 and 9) motifs (Fig. 1c). Apart from sharing conserved motifs, each subfamily members have unique motifs. Putative functions of these motifs are given in Additional file 3 Table S1. These results indicate that OsPYL members clustered in the same subfamily show similar motif characteristics, suggesting functional similarities among members, while presence of unique motifs might carry out unique/specialized biological functions.

Chromosomal distribution of OsPYLs across rice genome

OsPYL genes were found to be unevenly distributed across 12 rice chromosomes (Fig. 2). Rice chromosome 6 harbours 5 PYLs (OsPYL2, OsPYL7, OsPYL8, OsPYL9 and OsPYL13), chromosome 2 harbours 3 PYLs, (OsPYL3, OsPYL10 and OsPYL12), and chromosome 5 harbours 2 PYLs (OsPYL5 and OsPYL12). Six of the 12 chromosomes do not harbour PYL genes.

Fig. 2
figure 2

Chromosomal distribution of PYL genes on rice chromosomes. Chromosome numbers are showed at the top of each chromosome. The names of each OsPYL gene are shown on the right side of each chromosome. Subfamily I, II and III members are indicated in blue, green and red font, respectively. The bars on the chromosomes indicate the positions of the PYL genes. The figure was generated using Map Tool from Oryzabase

Full size image

Identification of PYL members in other species

To study the evolutionary relationships between rice OsPYLs and PYLs of other grass family members, amino acid sequence OsPYLs were used as queries to search genome of wheat, maize, brachypodium, sorghum, foxtail millet and barley (Additional file 4). We identified 26 TaPYLs as previously reported [49]. Nine TaPYLs were identified in each diploid genome except TaPYL2 which was absent in B genome. In maize, Brachpodium, foxtail millet, barley and sorghum 13, 10, 9, 9 and 8 PYLs, respectively, were identified. In OsPYL7, 8, 9 and 13 typical latch residues (HRL) are replaced by HML (Additional file 4).

Based on the phylogenetic analysis, PYLs could be broadly classified to 3 subfamilies. There are 29, 41 and 31 members in Subfamily I, II and III, respectively in the eight species analysed. Therefore, PYL subfamily I and II have the lowest and highest PYL members, respectively, in grass family (Fig. 3).

Fig. 3
figure 3

Phylogenetic analysis of PYL proteins from Arabidopsis (14 AtPYL), Brachpodium (9 BdPYL), Rice (13 OsPYL), Wheat (26 TaPYL), Maize (13 ZmPYL), Sorghum (8 SbPYL), Barley (9 HvPYL) and Foxtail millet (9 SiPYL) were used using the maximum likelihood method. The PYL proteins are classified into 3 subfamilies: I, II and III depicted by blue, green and red colour respectively

Full size image

Genome wide synteny analysis of PYL gene family

Gene duplication events including tandem and segmental duplications play an important role in broadening gene family during the evolutionary process [72]. Therefore, to gain insight into the genetic origins and evolution of the PYL gene family across six species, genome wide collinearity analysis was performed. Collinearity network grouped PYL genes across different taxas into five clusters where nodes represent individual PYL gene, while edges (lines between points) represent syntenic relationship amongst them (Fig. 4). Each cluster depicts high sequence similarity which might be a result of tandem gene duplication in the course of evolution. Interestingly, all PYL genes in cluster 1 belonged to subfamily II based on phylogenetic classification (Fig. 3). Notably cluster 3 comprising of PYL2 and PYL3 of different taxas was found to be least conserved as all the PYL genes form a closed interconnected network. On the other hand, PYL genes in cluster 1 were found to be most conserved. Further, we analysed whole genome duplication (WGD) events for PYL gene family between Arabidopsis and rice genomes by drawing whole genome Synteny blocks using CIRCOS (Fig. 5). Out of total 27 genes queried (14 Arabidopsis PYLs and 13 rice PYLs), only 20 PYL gene pairs formed collinearity blocks (11 Arabidopsis PYLs and 9 rice PYLs). Arabidopsis PYL genes were highly duplicated on chromosome two and four, while collinear gene pair between Arabidopsis and rice PYL genes was highest between Arabidopsis chromosome 2, 4, 5 and rice chromosome 1, 2 and 5. Some of the PYL genes exhibited multiple collinearity. We also found that collinearity of PYL4, PYL5 and PYL6 genes belonging to subfamily II was highest between the genome of two species. Moreover AtPYL2, and AtPYL3 of subfamily III, AtPYL12 of subfamily II, and OsPYL8, 9, 11 and 13 of subfamily I do not form any collinearity blocks (Additional file 5 Table S2). These results further fortify our findings that PYL genes belonging to subfamily II and III have been evolutionarily conserved, while genes of subfamily I are least conserved. Collinearity blocks at genome scale of Arabidopsis and rice was also constructed which showed gene duplication was high within the species (Additional file 6 Fig. S1). Further to infer extent of selection pressure in the divergence of PYL genes, the non synonymous (Ka) and synonymous (Ks) values were evaluated for the orthologous gene pairs (Table 2). A total of 65 ortholog pairs were formed for which the average Ka/Ks value was 0.070, suggesting that PYL family across the species were under purifying or stabilizing selection during evolution (Additional file 7).

Fig. 4
figure 4

Synteny network of PYL genes across six different plant species Nodes represent syntenic genes and edges (lines) represent a syntenic connection between two nodes. 181 homologous gene pairs exist among PYL from A.thaliana, B. distachyon, O. sativa, Z. mays, S. bicolor and H. vulgare at genome wide scale. Each cluster represents genes with high sequence similarity

Full size image
Fig. 5
figure 5

Circle plot showing collinearity in the PYL gene family. Collinearity in Arabidopsis and rice is highlighted with red curved lines over the gray background (genomic collinearity). Chromosomes are labelled in the format ‘species abbreviation’ + ‘chromosome ID’. At, Arabidopsis thaliana; Os, Oryza sativa

Full size image
Table 2 The Ka/Ks values of the homologous PYL gene family in Oryza sativa among genome of A.thaliana, B. distachyon, S. bicolor, Z. mays and H. vulgare
Full size table

Identification of miRNAs targeting OsPYL genes in rice

In order to predict miRNAs that may target OsPYLs, the cDNA sequences of OsPYL genes of rice were used as input in psRNATarget [73] against all the rice mature miRNAs available in miRbase [74] (Additional file 8). Total of 32 unique potential miRNAs targeting the OsPYL family members of rice were identified with mature miRNAs of 21–23 nucleotide long, Watson-Crick or G/U base pairing and stable minimal folding free energy (MFE). Some of the miRNAs were found to target specific subfamily. For example osa-miR5832 targets OsPYL1-OsPYL3 of subfamily III, while osa-miR5075 targets OsPYL4 and OsPYL5 of subfamily-II and OsPYL3 of subfamily I. Two members of osa-miR5157-3p family (osa-miR5157a-3p and osa-miR5157b-3p) were predicted to target OsPYL8, OsPYL9 and OsPYL10 of subfamily I. The miRNAs that potentially target OsPYLs ranged from minimum one (OsPYL4) to maximum of eight (OsPYL2) (Fig. 6a). The prominent inhibitory action by most of the miRNAs predicted to target OsPYL members across the three subfamilies was through cleavage (Additional file 8). Interestingly, most of the miRNAs were found to play a key role in stress responsiveness and development (Additional file 8). Microarray expression analysis of the identified miRNAs showed that majority of them are downregulated in different tissues and under stress conditions, while osa-miR820a and osa-miR408-3p targeting OsPYL1 and OsPYL6, respectively, showed the highest expression level in all tissues and under different abiotic stresses (Fig. 6b). Thus, regulation of OsPYL genes through miRNA mediated sequence specific interaction might play a decisive role for plants to respond to growth and environmental stimuli.

Fig. 6
figure 6

Identification potential miRNAs targeting PYL genes. a Schematic representation of targeted regulatory relations between miRNAs and their target PYLs using Cytoscape. Black line represents the interaction, and the blue and yellow box represents the miRNAs and its target OsPYL genes, respectively. b In silico expression analysis of putative PYL-targeting miRNAs. Log TPM (Transcript per million) values calculated after adding pseudocount of one to each miRNA count (Heat map generated by PmiRExAt http;//pmirexat.nabi.res.in/). c Different coloured circle, triangle and star represent PYL genes belonging to subfamily I, II and II respectively, targeted by the corresponding miRNAs

Full size image

Identification of SNPs in OsPYL members

To get a deeper insight into allelic variation of OsPYL members across 12 rice varieties selected based on stress responses [75]. Rice SNP-Seek database [76] (https://snp-seek.irri.org/) was queried SNPs in OsPYL genes using Nipponbare reference genome. Among 13 PYL genes in rice, non-synonymous SNPs were identified in 10 OsPYLs, but not in OsPYL1, OsPYL5 and OsPYL10 (Table 3). This shows that PYL1, PYL5 and PYL10 genes are highly conserved across different rice genotypes. Interestingly, on protein structural basis, we found that majority of the Single Amino acid Polymorphisms (SAPs) were located across the helix grip fold domains, while some were located on CL loops surrounding ABA binding pocket [20, 23] (Additional file 9).

Table 3 Single Amino acid Polymorphisms (SAPs) in PYL proteins of selected rice genotypes
Full size table

Identification of cis-regulatory elements (CREs) in promoters of OsPYLs

Rice, being a sensitive crop to various biotic and abiotic stresses, needs to adapt swiftly to frequently changing stresses [66]. The control of gene transcription via CREs in the promoter remains a pivotal mode of regulation of gene expression. To investigate the potential CREs in OsPYL gene family in rice, the promoter sequences of approximately 2.0-kb upstream from the translation start sites of individual PYL receptors, were taken from RGAP [68] and searched against New PLACE [77] (https://www.dna.affrc.go.jp/PLACE/?action=newplace). A total of 193 putative CREs were predicted across thirteen OsPYL genes with a range from 62 to 105 CREs (Additional file 10). The prevalence distribution of 20 key CREs are schematically depicted (Fig. 7). Some of these elements are conserved across OsPYL family, and might be critical in imparting stress and developmental regulation. We have identified certain CREs that can be functionally attributed to individual OsPYL members or subfamily. For an example OsPYL8 and OsPYL9 has endosperm and seed specific CREs like -300CORE [78], and 2SSEEDPROTBANAPA [79]. Previously these elements have been reported to be associated with endosperm specific activity of OsPYL8 and OsPYL9 [46]. Likewise ABREZMRAB28 is induced by ABA and is a binding site for CBFs [80]. It has been recently reported that overexpression of OsPYL10 imparts dehydration and freezing tolerance in transgenic rice [47]. We have identified cis-element TAAAGSTKST1 [81] which is a target site for transcription factor governing guard cell specific gene regulation in stomata. All the identified CREs have been grouped into four major categories based upon function and their response to stimuli (Fig. 8). In this study, hormone responsive elements like abscisic acid responsive elements (ABRE) form the major proportion of CREs. Interestingly gibberellic acid responsive elements (GARE) and auxin responsive elements (ARE) were also found in abundance in the promoters, suggesting potential hormonal cross talk at the expression level of ABA receptors (Fig. 8a). Amongst stress responsive elements, dehydration responsive elements (DRE) forms the major group followed by low temperature responsive elements (LTRE) (Fig. 8b). On the basis of metabolic functions, the proportion of amylose/starch responsive elements (34%) followed by carbohydrate responsive elements (27%) was notably highest, while elements involved in amino acid metabolism comprised least (6%) of the CREs identified (Fig. 8c). The proportion of elements that might be involved in photosynthetic machinery and light responsive elements (LRE) was found to be highest (34%) followed by seed and endosperm specific elements (19%) (Fig. 8d). All OsPYL promoters had more than one stress-response-related CREs. CREs associated with hormonal regulation, including ABRE, AuxRR-core, CGTCA motif, P-box, TCA-element, and TGA-element were identified in most of the OsPYL genes promoter. TC-rich repeats, which are involved in low temperature, drought inducibility and defence responsiveness, were also found in many OsPYLs (Additional file 11). This suggests that OsPYL genes are regulated by diverse development and stress responses.

Fig. 7
figure 7

cis-regulatory Elements (CREs) in the promoter of PYL genes. Positional distribution of predicted CREs on OsPYL promoters are shown as vertical bars. Promoter sequences (− 2000 bp) of thirteen OsPYL genes were analyzed by using NewPLACE. Legend depicting the colour of individual cis elements

Full size image
Fig. 8
figure 8

Percentage wise distribution of cis-regulator elements (CREs) in the promoters of OsPYL genes based upon the putative functions. a Hormone responsive CREs. b Stress responsive CREs. c Metabolic responsive CREs. d Growth and biological process responsive CREs

Full size image

In silico expression analysis of OsPYL genes

Spatiotemporal and stress responsive expression analysis of OsPYL genes was carried out using GENEVESTIGATOR database [82] (https://genevestigator.com/gv/). OsPYL1, OsPYL2, OsPYL6, OsPYL10 and OsPYL11 showed expression in most tissues across developmental stages (Fig. 9a and b). OsPYL7, OsPYL8 and OsPYL9 were found to be specifically expressed in endosperm and embryo suggesting their potential role in seed development in rice. In response to rice blast fungus (Magnaporthe oryzae) infection, OsPYL5 and OsPYL6 showed upregulation in leaf of indica cv. Pusa Basmati 1. Bacterial leaf streak pathogen (Xanthomonas oryzae pv. oryzicola) inoculation also induced these two PYLs and OsPYL12 (Fig. 10a). Ethylene moderately upregulated only OsPYL5 (Fig. 10b). Alkali treatment induced the expression of OsPYL6 to > 1.5 folds in leaves Drought stress induced the expression of PYL1, PYL8 and PYL10 by > 1.5 fold. Interestingly, drought stress upregulated the expression of PYL10 by > 1.5 fold leaf but it was downregulated in the roots (Fig. 10c). Heat stress upregulated OsPYL1 and downregulated OsPYL6 in leaf. Both drought and salt stresses downregulated OsPYL5 and OsPYL6 (Fig. 10c).

Fig. 9
figure 9

Expression potential of PYL genes in different tissues and developmental stages of rice. a Developmental stages. Stage development shown in the picture are Germination, seedling; tiller initiation; stem elongation, booting, heading, anthesis, milk stage of grain development, dough stage of grain development and mature grain stage (top panel). b Different tissues. Expression analysis was carried out with mRNAseq datasets using Genevestigator

Full size image
Fig. 10
figure 10

Abiotic stress regulated expression of PYL genes in rice. Expression analysis was carried out with mRNAseq datasets using Genevestigator. a Biotic stress. b Ethylene. c Abiotic stresses. The colour scale represents Log2 of average signal values

Full size image

Real time qRT-PCR expression profiling of OsPYL genes in different tissues

Tissue and stress responsive expression of PYL genes were analysed to understand their role in development and stress tolerance. OsPYL8 and OsPYL9 showed with highest expression in seeds, while OsPYL1, OsPYL11 and OsPYL13 showed highest expression in panicle among the tissues (Fig. 11). Among the PYLs, OsPYL2 showed highest expression in roots of the plants at reproductive stage as compared with that in other tissues. Interestingly, many PYLs (OsPYL1, OsPYL4, OsPYL5, OsPYL8, OsPYL9, OsPYL11, OsPYL12 and OsPYL13) also showed high levels of expression in stem tissue at reproductive stage as compared with that in seedling roots (Fig. 11).

Fig. 11
figure 11

Real-time qRT-PCR expression analysis of tissue specific expression of OsPYL genes in rice. Tissue specific expression of OsPYLs was analyzed by qRT-PCR in seed, root and shoot at seedling stage, and root, stem, flag leaf and panicle at reproductive stage of indica rice cv. Nagina22. The expression level in the root of seedling was taken as calibrator (1) and the fold change was analyzed via the 2-ΔΔCT method using the rice Ubiquitin gene as an internal control. Values represent the mean + SD of three biological replicates

Full size image

Real time qRT-PCR expression profiling of OsPYL genes in responses to abiotic stresses

All the subfamily III OsPYLs were significantly upregulated by all the abiotic stresses at least in one tissue at seedling stage in drought tolerant rice cv. Nagina 22 (Fig. 12). OsPYL1 was significantly upregulated in both root and shoot at seedling stage by abiotic stresses except NaCl which downregulated its expression in root (Fig. 12). Similarly contrasting tissue specific regulation was observed for OsPYL2 under osmotic (PEG) stress and ABA, where it was upregulated in shoot and downregulated in root at seedling stage. OsPYL3 was upregulated only in shoot by PEG stress, while it was upregulated both in root and shoot by NaCl and heat stress, and downregulated in the both the tissue by cold stress (Fig. 12). The subfamily II OsPYLs (PYL4, PYL5 and PYL6) were mostly downregulated both in root and shoot by all stresses and none were upregulated by stress at seedling stage (Fig. 12). Among subfamily I OsPYLs (PYL7-PYL13), OsPYL8, OsPYL9 and OsPYL13 were significantly upregulated by osmotic stress (PEG) in shoot. Salt stress significantly upregulated OsPYL7, OsPYL8 and OsPYL11 in shoot, and OsPYL8 and OsPYL9 in root. Cold stress significantly upregulated OsPYL7, OsPYL9 and OsPYL11 in shoot, and OsPYL13 in both root and shoot at seedling stage. Heat stress significantly upregulated OsPYL8, OsPYL9 and OsPYL10 in both root and shoot, and OsPYL11 and OsPYL12 only in shoot at seedling stage (Fig. 12). ABA regulated the expression of all OsPYLs except OsPYL9, OsPYL11 and OsPYL13 at least in one tissue at seedling stage in rice ABA significantly upregulated OsPYL2, OsPYL7 and OsPYL12 in shoot, and OsPYL1, OsPYL8 and OsPYL10 in root at seedling stage (Fig. 12).

Fig. 12
figure 12

Real-time qRT-PCR expression analysis of OsPYL genes under abiotic stresses at seedling stage in rice. Total RNA isolated from 14 days old seedlings of rice cv. Nagina22 treated with osmotic stress (PEG), salt, cold, heat and ABA treatments for 6 h and control plants were used for qRT-PCR analysis. The expression level in control sample was taken as calibrator (1) and the fold change was analyzed via the 2-ΔΔCT method using the rice ubiquitin gene (UBQ) was used as an internal control. Values represent the mean + SD of three biological replicates

Full size image

At reproductive stage, tissue specific regulation of PYLs was observed under drought stress. In root tissue, drought stress significantly upregulated the expression of OsPYL4, while it downregulated rest of the OsPYLs except OsPYL12. In contrast, in the stem tissue, drought stress significantly upregulated most OsPYLs, except OsPYL7 and OsPYL13 which were downregulated, and OsPYL10 which was unaltered. Drought stress significantly upregulated OsPYL2 and OsPYL4 in flag leaf and OsPYL8, OsPYL9 and OsPYL13 in panicles (Fig. 13). Thus, at reproductive stage, drought stress either up- or down-regulated the expression all the OsPYLs in most of the four different tissues examined (Fig. 13).

Fig. 13
figure 13

Real-time qRT-PCR expression analysis of OsPYL genes under drought and heat stress at flowering stage in rice. Total RNA isolated from rice cv. Nagina22 at anthesis stage was subjected to drought (− 80 kPa) and heat stress, and control plants were used for qRT-PCR analysis. The expression level in control sample was taken as calibrator (1) and the fold change was analyzed via the 2-ΔΔCT method using the rice ubiquitin gene (UBQ) was used as an internal control. Values represent the mean + SD of three biological replicates

Full size image

Heat stress at reproductive stage differentially regulated the expression of all OsPYLs in vegetative tissues (root, stem, leaf) except that of OsPYL1 in stem, while in panicle it regulated most PYLs except OsPYL7, OsPYL10 and OsPYL12. Among the subfamily III members, OsPYL3 was significantly upregulated under heat stress in all four tissues (Fig. 13). Among the subfamily II members, OsPYL6 was downregulated in all four tissues, OsPYL4 was upregulated in stem and leaf but downregulated in root and panicle, and OsPYL5 was downregulated in roots and upregulated in rest of the three tissues under heat stress (Fig. 13). Among the subfamily I members, OsPYL8, OsPYL9, OsPYL10 and OsPYL12 were significantly upregulated in both root and stem, OsPYL7, OsPYL8 and OsPYL9 in flag leaf and OsPYL13 in panicle under heat stress (Fig. 13).

Discussion

Gene structure and evolution

ABA is a key phytohormone that governs various plant development and stress response processes. ABA is perceived by PYL family of receptors, which are the largest plant hormone receptor family known [12]. Despite arduous efforts investigation on PYL gene family has largely been confined to characterization of PYL genes mainly in Arabidopsis, while only limited efforts have been made on genome wide characterization of PYL gene family for elucidating their role in multiple stresses and their evolutionary relationship in major crops. In the present study, a genome wide comprehensive analysis of PYL genes in rice and its potential role in development and abiotic stress responses was investigated using bioinformatic and experimental approaches. A total of 13 OsPYLs were identified from rice genome using Arabidopsis PYL protein sequence as queries as previously reported [44]. The sequences of all 13 OsPYL genes from upland indica rice variety Nagina22 were used in the present study. Based on phylogenetic relationship with Arabidopsis, OsPYL family can be broadly classified into three subfamilies: I, II and III (Fig. 1a). Subfamily II and III PYLs of rice and Arabidopsis are intronless, while all seven members of OsPYLs belonging to subfamily I have introns (Fig. 1b). The organizations of intron/exon and the number of exons in the surveyed rice ABA receptors were similar to those orthologs in maize, tomato, rubber, cotton, brachypodium and sorghum [54, 56, 59, 61, 63, 83]. These results imply that the exon/intron organizations of OsPYLs are closely related to the phylogenetic relationship of the genes (Fig. 1a). Introns play key role in post-transcriptional regulation gene expression by splicing-dependent and splicing-independent intron-mediated enhancement of mRNA accumulation [84]. All the PYL members of subfamily I has evolved introns to fine tune their regulation.

To further understand the diversification of PYL gene family at protein level, ten conserved motifs were acquired for each protein using MEME Suit (Fig. 1c). All the PYL proteins had motif 1 that has the conserved Gate and Latch domain of PYL receptor [23, 28]. Motif 2 and motif 3 were present in all PYL members except the non-functional OsPYL7 and OsPYL12. HHpred analysis to affirm if the motifs obtained from the MEME analysis are similar to any of the known protein motifs revealed that Motif1, 2 and 3 belongs to Polyketide cyclase/dehydrase and lipid transport superfamily protein. It was observed that these novel motifs did not show any significant similarity with the known motifs (Additional file 3). This indicates that all the identified OsPYLs have typical subfamily features and the proteins classified into the same subgroup share similar protein motifs.

ExPaSy analysis of physical properties of the OsPYLs from indica rice cultivar Nagina 22 showed that except OsPYL7, OsPYL12 and OsPYL13, all other receptors had similar protein length and physical properties (Table S1). Amino acid sequence of most ABA receptor were quite similar between japonica and indica baring few SNPs in OsPYL2, OsPYL3 and OsPYL9 (NCBI Accession no. KJ634482, KM371729, KM371729). Compared to japonica rice, OsPYL3 in indica rice has an insertion of nine nucleotides, increasing the amino acid sequence by three residues.

Chromosomal distribution of PYL genes across 12 rice chromosomes showed uneven distribution of PYLs with as many as five PYLs located on six chromosomes. OsPYL8 and OsPYL9 having 95.5% sequence similarity at nucleotide levels are located in nearby positions on chromosome 6 suggesting a tandem gene duplication event that might have caused evolution of one these receptors. Unequal number of PYLs across seven different plant species ranging from 8 to 14 (sorghum and Arabidopsis) identified in the current study.

We analysed ABA receptors in seven members of grass family. Phylogenetic analysis of PYL protein sequences from rice, maize, sorghum, barley, brachypodium, wheat and foxtail millet showed that PYL gene family across seven species can be broadly grouped into three subfamilies similar to that in Arabidopsis (Fig. 3). Subfamily II has the maximum number of PYL members (41), followed by Subfamily III (31 PYL genes) and Subfamily I has the least members (29 PYL genes). These members of grass family analysed here have evolved from a common ancestor about 96 Ma, and BOP (Bambusoideae-Oryzoideae-Pooideae) and PACCMAD (Panicoideae, Arundinoideae, Chloridoideae, Centothecoideae, Aristidoideae, Danthonioideae) clades diversified about 70 Ma. Arabidopsis and rice which diverged from a common ancestor about 120–200 Ma have similar number of PYLs. It is interesting to note that rice (BOP clade) and maize (PACCMAD clade) have 13 PYLs each, while other members of BOP (wheat – each diploid genome, barley and Brachypodium) and PACCMAD (sorghum and foxtail millet) have only 8–9 PYLs, respectively. The sub-family III of all seven members of grass family analysed here have 3 PYLs (PYL1-PYL3), while it varied among grasses in sub-family II (4–7 PYLs) and family I (2–7 PYLs). The conservation of three subfamilies albeit with different number of members suggests non-redundant roles for each subfamily.

Collinearity results showed that approximate 181 (Additional file 12 Table S3) homologous gene pairs existed among PYLs from A. thaliana, B. distachyon, O. sativa, Z. mays, S. bicolor and H. vulgare at genome wide scale (Fig. 4), grouping PYL genes into 5 clusters with each cluster depicting high sequence similarity and might therefore share same functional domains. Surprisingly PYLs in cluster 5 belonged to subfamily III to phylogenetic classification. Synteny analysis of PYL family between Arabidopsis and rice showed that collinearity blocks between PYL members of subfamily II were highest, while members of subfamily III formed least collinearity pairs (Fig. 5). These results suggest that PYL family expanded through segmental duplication events during evolution. The evolutionary history subfamily II members might provide more clues to the origin and evolution of the PYL gene family.

Non synonymous SAPs in OsPYLs identified from 3 K SNP seek database showed that frequency of SAPs was high across indica rice cultivars as compared to landraces (Table 3). Moreover most of the SAPs were present across START domain that might modulate the ABA binding ability of ABA receptors in response to different types and magnitude of stresses.

Regulation of gene expression

We carried out a systematic analysis of CREs in promoter regions of PYLs and identified various types of CREs such as stress responsive elements, hormone responsive elements, metabolic responsive elements and elements involved in growth and development (Additional file 10). The number of ABA and stress responsive elements was found to be highest in the promoters across OsPYL genes. Most OsPYL promoters contain CREs stress signalling and pathogen response. Each promoter of OsPYL genes possessed more than one cis elements involved in photosynthesis and light responsiveness, while elements involved in carbohydrate and starch metabolism was equally high across all promoters. This suggests that OsPYL genes might be regulated by photosynthesis and carbohydrate metabolism signalling. The knowledge of presence of regulatory elements in the promoters of PYL genes can further help in functional characterization and tailoring PYL receptors in rice and other commercially important crops.

We identified 32 candidate miRNAs that may potentially target OsPYLs mRNA to regulate their expression in rice (Fig. 6a). Most of these miRNAs identified have been implicated in stress responsiveness and development (Additional file 8). In an earlier study with Brassica napus, 26 miRNAs targeting 11 BnPYL genes were identified, and predicted that 10 members of miR169 target BnPYL1–4 [85]. For better understanding, each OsPYL gene targeted by different miRNAs is depicted by different colour and shape (Fig. 6c). In our study, many PYLs were targeted by multiple miRNAs with maximum of 8 different miRNAs targeting OsPYL2. Although OsPYL8 and OsPYL9 have very high sequence similarity, OsPYL9 is targeted by 7 miRNAs while OsPYL8 is targeted by only 4 miRNAs. The miR5075 targets five OsPYL genes and thus may play a critical role in fine tuning the expression of PYLs. In silico expression analysis of the identified miRNAs under different tissues and abiotic stresses revealed that apart from osa-miR820a and osa-miR408-3p, other miRNAs were downregulated (Fig. 6b). It is important to note that ABA-activated SnRK2 kinases interact with and phosphorylate SERRATE (SE) and HYPONASTIC LEAVES (HYL1) proteins involved in miRNA biogenesis [86]. The predicted miRNA mediated regulation of PYLs suggests that miRNA mediated regulation of PYLs may be important not only for stress response of rice but may also play a key role in feedback regulation of overall miRNA biogenesis through PYL-mediated regulation of SnRK2.2/3/6. Further functional characterization of the predicted miRNAs would enable us to better understand the regulatory mechanism underlying ABA receptors and miRNA biogenesis.

Analyzing the spatiotemporal pattern of gene expression across the broad spectrum of different tissues, developmental stages and stress conditions would provide insight into the physiological and developmental functions of OsPYLs. In silico as well as q-RT-PCR expression analysis of OsPYLs different stress treatments and developmental stages showed that PYL genes are regulated in a tissue and developmental dependent manner and by multiple stresses (Fig. 10, 11, 12). Two members of subfamily II (OsPYL5 and OsPYL6) and all 3 members of subfamily III showed higher expression potential in different tissues across developmental stages, while only OsPYL10 and OsPYL11 among the 7 members of subfamily I showed higher expression potential in different tissues across developmental stages (Fig. 9). This suggests that these PYLs have multiple roles throughout the growth and development of rice. The PYL8 and PYL9 orthologs in Arabidopsis have been shown to play important role in lateral root formation during seedling growth [5]. Interestingly, OsPYL7, OsPYL8 and OsPYL9 showed expression during germination stage and seed development (embryo, endosperm and caryopsis) (Fig. 9). Real-time qRT-PCR analysis also showed that OsPYL8 and OsPYL9 were highly expressed in panicles and seeds (Fig. 11). In a previous study also it was found that these two PYLs were highly expressed in seeds [42]. These results suggest that OsPYL7, OsPYL8 and OsPYL9 may have specific role in germination as well geed development. Although OsPYL7 was predicted as non-functional due to lack of C-terminal sequences (motif2), it was found to be co-expressed with OsPYL8 and OsPYL9 in seeds (embryo, endosperm and caryopsis) (Fig. 9). Further studies may illuminate whether OsPYL7 interact OsPYL8 and OsPYL9 to regulate rice seed development. In our qRT-PCR expression analysis also OsPYL8 and PYL9 showed very high levels of expression in panicle and seeds as compared with other tissues (Fig. 11). Notably we also identified CREs 2SSEEDPROTBANAPA and − 300 CORE, which are involved in seed and endosperm specific expression, specifically present in promoter region of OsPYL8 and OsPYL9. This further strengthens the proposed roles of OsPYL8 and OsPYL9 in seed and endosperm specific activity. OsPYL5, OsPYL9, OsPYL11 and OsPYL13 may play an important role in regulation of source activity as their expression was higher in flag leaf which contributes to > 70% of current assimilation for grain development.

A previous study showed that only OsPYL6 (their nomenclature, OsPYL4) was upregulated, while OsPYL2, OsPYL3, OsPYL4 and OsPYL10 were downregulated by 200 μM in 14 days old seedlings of japonica cv. Nipponbare [42]. In contrast, our analysis showed that 100 μM ABA significantly upregulated OsPYL1, OsPYL8 and OsPYL10 root, and OsPYL1, OsPYL2, OsPYL5, OsPYL7 and OsPYL12 in shoots of drought tolerant indica rice cv. Nagina22 (Fig. 12). In consistent with Tian et al. [42], in our study also ABA downregulated OsPYL2, OsPYL3 and OsPYL4 roots, OsPYL4 and OsPYL10 in shoots. Thus, ABA-mediated regulation of OsPYLs appears to be genotype and tissue dependent.

Previous functional validation studies have shown that constitutive/stress-inducible overexpression of OsPYL2 [42], OsPYL10 [42, 47, 87], and OsPYL11 (=RCAR5) [44] conferred tolerance to abiotic stresses. In a previous study, expression analysis of OsPYL11 (=RCAR5) [44] under salt, PEG and ABA treatments showed that it is significantly downregulated by ABA and salt stress. In our analysis PEG downregulated the expression of OsPYL11 in root, while salt stress upregulated in shoots of drought and heat tolerant cv. Nagina 22. In a previous study, OsPYL10 [47] expression was found to be downregulated by PEG, NaCl and cold stresses in the roots, but was found to be upregulated by ABA in roots. In this study also similar expression of OsPYL10 was found under these treatments. However, in none of the previous studies expression of all OsPYLs under different stresses were examined.

In this study, qRT-PCR expression analysis showed that all the OsPYLs are regulated by one or more abiotic stresses (osmotic/PEG, drought, salt, heat and cold) in at least one tissue/development stage of rice plant (Fig. 12, 13). This suggests that all PYLs play important roles in abiotic stress responses of rice. In general subfamily I and III PYLs were upregulated by different abiotic stresses and ABA at seedling stage of drought and heat tolerant rice cv. Nagina 22, while subfamily II PYLs were downregulated (Fig. 12, 13). This suggests that subfamily-wise stress response role at seedling stage.

In consistent with the presence of CCAATBOX1 [88] CRE that act as heat responsive elements in the Promoters of PYLs, heat stress upregulated OsPYLs except OsPYL6 and OsPYL11, at least in one tissue (Fig. 13). Over all, drought stress downregulated all PYLs except OsPYL4 which was upregulated and OsPYL12 which was unaltered, while heat stress upregulated OsPYL1, OsPYL3 OsPYL8, OsPYL9, OsPYL12 and OsPYL12 in the roots at reproductive stage (Fig. 13). This suggests contrasting regulation and function of OsPYLs in roots under drought and heat stress. Both drought and heat stress upregulated OsPYL2, OsPYL3 OsPYL4, OsPYL5, OsPYL8, OsPYL9 and OsPYL12 in the stem at reproductive stage, while OsPYL1, OsPYL6 and OsPYL11 were upregulated only by drought and OsPYL10 was upregulated only by heat in the stems (Fig. 13). Interestingly all the three OsPYLs (OsPYL2, OsPYL4 and OsPYL13) upregulated in flag leaf in response to drought stress were also upregulated under heat stress. In the panicle only OsPYL13 was commonly upregulated by both drought and heat at reproductive stage. OsPYL8 and OsPYL9 were specifically upregulated by drought but were downregulated by heat in panicles, while OsPYL3 which was upregulated under heat but was downregulated under drought stress in panicle (Fig. 13). The diverse expression patterns of OsPYLs were indicative of their functional distinctiveness.

Conclusion

The present study is a comprehensive functional identification and characterization of PYL gene family in indica rice at genomic level. Evolutionary relationship of PYL genes in rice and other cereal crops was established which grouped PYL genes into three subfamilies that are structurally and functionally evolutionarily conserved. Identified cis elements could help in understanding the diversified role of PYL receptors in response to different stresses and developmental stages. These data will provide the basis for understanding evolutionary history and the developmental roles of OsPYL genes in rice, and may be helpful for future exploration of the biological functions of OsPYL genes. These findings will also serve to extend our knowledge for identifying candidate genes that improve plant architecture under stress conditions and enable potential breeding and genetic improvements for other agriculture crops.

Methods

Identification of PYL genes from plant species

To identify ABA receptors of different species, Arabidopsis PYLs were used as query in EnsemblPlants database (https://plants.ensembl.org/index.html) against their respective species genome with a threshold of 10− 4 and Match/Mismatch score of 2 and − 3.

Chromosomal distribution of PYL genes in rice

Chromosomal location of OsPYL genes was determined with respect to their position and information retrieved from rice genome sequences. The physical map information on chromosome number, length and gene loci were obtained from rice genome annotation project (RGAP) (http://rice.plantbiology.msu.edu/). Elementary physical map depicting the location and distribution of OsPYL gene family was drawn using Map Tool software from Oryza base (https://shigen.nig.ac.jp/rice/oryzabase/) with default parameters.

Sequence retrieval and phylogenetic analysis

Gene sequences of rice PYLs were identified from the Rice Genome Annotation Project [68] using Arabidopsis PYL protein sequences from Arabidopsis Information Resource [89]. In the present study, Nagina22 sequence for OsPYL7 and OsPYL13 were retrieved from Rice SNP-Seek Database (https://snp-seek.irri.org/), while rest of the PYLs were coned and sequenced from Nagina 22. Sequences of PYLs from Brachypodium, Sorghum, barley, maize, foxtail millet and wheat genomes were retrieved from EnsemblPlants database (https://plants.ensembl.org/index.html). For phylogenetic analysis, amino acid sequences of putative PYL proteins of H. vulgare, S. bicolor, O. sativa and A. thaliana, T. aestivum, Z. mays, S. italica and B. distachyon were analyzed. The genes of PYLs were named based on numbering and sequence homology with A. thaliana orthologs genes. Multiple sequence alignment was executed by ClustalW 2.0 program [90]. Phylogenetic trees were constructed using MEGA X [70] by the maximum likelihood method [69].

Analysis of gene structure, conserved motif and protein properties

Conserved motifs were also predicted for all 13 OsPYL proteins using MEME Suite v5.1.0 [91, 92]. Functional annotations of these motifs were performed using HHpred (http:// toolkit.tuebingen.mpg.de/hhpred) [93]. Maximum number of motifs was specified as 10. Parameters on minimum/maximum width were specified as 6 and 50, while the range for minimum and maximum sites per motif were kept as 2 and 13, respectively. The motifs were serially numbered according to their frequency of occurrence in MEME. Motifs were placed adjacent to their respective OsPYLs in accordance with their subfamily signatures based on phylogenetic relationship. Using the ExPASy database, the isoelectric points and molecular weights of the OsPYLs were predicted.

miRNA identification and in silico expression analysis

miRNA targeting PYL genes in rice were predicted using psRNATarget [73] against all the rice mature miRNAs that were reported in miRbase [74] and the network was created using Cytoscape [94]. In silico expression analysis of identified miRNAs was carried out using miRid as query against rice datasets of plant miRNA expression atlas database PmiRExAt (http://pmirexat.nabi.res.in/index.html) [95].

Genome wide collinearity and Ka/Ks analysis of PYLs

Analysis of homologous gene pairs of PYLs among Arabidopsis thaliana, Oryza sativa, Zea mays, Brachypodium distachyon, Sorghum bicolor and Hordeum vulgare at genome level was carried out. Whole genome primary transcript file of each species was obtained from Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) and whole genome reciprocal protein to protein BLAST in all pair wise combinations (36 permutation combinations) was carried out. BLAST result of all the possible combinations was merged. Annotation file of all selected species were imported into Cytoscape and collinearity network was constructed. Similarly for Synteny block calculation of PYL gene family between Arabidopsis and rice at genome scale were used. PYL genes were filtered form whole genome collinearity data along with gene positional information of gene was detected using ‘collinearity with gene families’ were queried in MCScanX and visualized in CIRCOS [96, 97] using default parameters. The values of nucleotide substitution parameter Ka (non-synonymous) and Ks were calculated for PYL gene family in Oryza sativa. Orthologs of rice PYL gene family were noted from collinearity blocks table and calculated using MCScanX software [96]. Pairwise global alignment for all ortholog pairs (protein sequences) was done online by EMBOSS Needle Pairwise Sequence Alignment tool [98]. Online program PAL2NAL [99] was used to convert protein sequence alignment and the corresponding mRNA sequences into a codon alignment and calculating Ka/Ks value from the aligned codon.

Identification of SNPs in PYL genes

Amongst available 3024 accessions, sequence of 13 PYL genes from 12 mega rice varieties comprising of contrasting genotypes in terms of abiotic stress sensitivity were fetched from Rice SNP-Seek database [76] against the reference Nipponbare sequence. Non-synonymous SNPs that could particularly translate to change in the protein sequence were identified across the selected genotypes. Domain based localization of SAPs was done on the basis of secondary structure of OsPYL proteins.

Identification of putative cis-regulatory elements (CREs) in the promoters

The 2000 bp upstream sequences from the translation start site of all of the OsPYL genes were obtained from Rice genome annotation project database. The putative cis-acting regulatory elements in these sequences were predicted using the NewPlace web server [77] and then to identify the putative CREs. Functional annotation of individual CRE was manually curated from place.seq (https://www.dna.affrc.go.jp/PLACE/place_seq.shtml); (Additional file 10).

Expression analysis of OsPYLs in rice

In silico expression analysis of ABA receptors at different developmental stages and stresses were analyzed using GENEVESTIGATOR database (https://genevestigator.com/gv/) [82]. The rice genotype Nagina 22 (Oryza sativa ssp. indica cv. Nagina 22) seeds, from our lab in Division of Plant Physiology, ICAR-IARI, New Delhi, was used for analysis of tissue specific and stress responsive expression of 13 OsPYL genes at seedling stage (14 day old) and reproductive stage using real time qRT-PCR. OsPYL expression was analyzed in different tissues from plants at anthesis stage exposed to control, drought and heat stress treatments. At seedling stage, plants grown in Yosida’s medium (YM) were treated with YM supplemented with 20% PEG 6000 (− 0.49 MPa), 200 mM NaCl (− 1.01 MPa; 20 dS/m) and cold (4 °C) stresses, Heat (42 °C) and 100 μM ABA for 6 h. Samples were collected from control, and treated plants and frozen in liquid nitrogen. Drought and heat stress was imposed at anthesis stage in Nagina22 grown in pot at green house, IARI, New Delhi conditions of 30 + 2 °C for control and drought while temperature of 42 + 2 °C for heat at a relative humidity (RH) of ~ 60–70%. Drought was imposed at reproductive stage by with-holding water till the soil matric potential (SMP) reached up to -80 kPa.

Quantitative real-time RT-PCR

Total RNA was extracted using RNeasyMini Kit (Qiagen, Germany) following manufacturers protocol and treated with DNaseI. Quantification of RNA was carried out using NanoDrop (Thermo Fisher, US). cDNA was synthesized from 2 μg of total RNA using Superscript III Reverse Transcriptase (Invitrogen). Real-time PCR was performed with Hotstart SYBR Green master mix (KAPA SYBR FAST; Universal). PCR conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 40 s in StepOne Real-Time PCR system (Applied Biosystems). The relative expression levels of OsPYL genes were calculated based on the comparative Ct method using the 2^ΔΔCt method [100] and all expressions were normalized against the Ubiquitin5 gene [101]. Root tissue ΔCt of seedling stage was used as calibrator for tissue specific expression analysis, while expression level control tissue (ΔCt) was used as calibrator for stress responsive expression analysis. The primers used are listed in (Additional file 13 Table S4).

Statistical analysis

The presented values are the means ± SE of three different experiments with three replicated measurements. Unpaired t-Test was used to compare significant differences based on Fisher’s LSD test at significance levels of P < 0.05 and P < 0.01 using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA).

Availability of data and materials

The sequences of ABA receptors cloned from rice cv. Nagina 22 were deposited in the NCBI and are available in the NCBI (https://www.ncbi.nlm.nih.gov/nucleotide/; Table 1; Additional file 1). The rice genome sequences, the physical map information on chromosome number and length and gene loci are available in RGAP (http://rice.plantbiology.msu.edu/), the Nagina22 sequence for OsPYL7, OsPYL12 and OsPYL13, the sequence of thirteen PYL genes from twelve mega rice varieties, the reference Nipponbare sequence are available SNP-seek (https://snp-seek.irri.org/, Table 1, Additional file 1), and the sequences of PYLs from Arabidopsis (https://www.arabidopsis.org) Brachypodium, Sorghum, barley, maize, foxtail millet and wheat genomes (https://plants.ensembl.org/index.html) are available in the respective database. The accession number and web links for the PYLs of these species are given in Additional file 4.

Abbreviations

ABA:

Abscisic acid

MW:

Molecular weight

NJ:

Neighbor-joining

ORF:

Open reading frame

pI:

isoelectric point

PYL:

PYR1-Like

PYR:

Pyrabactin Resistance

RCAR:

Regulatory Component of ABA Receptor

SNP:

Single nucleotide polymorphism

SAP:

Single amino-acid polymorphism

CRE:

cis regulatory elements

References

  1. Zhu JK. Abiotic stress signalling and responses in plants. Cell. 2016;167(2):313–24.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Nambara E, Marion-Poll A. Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol. 2005;56:165–85.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR. Abscisic acid: emergence of a core signalling network. Annu Rev Plant Biol. 2010;61:651–79.

    CAS  PubMed  Article  Google Scholar 

  4. Lumba S, Cutler S, McCourt P. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions. Annu Rev Cell Dev Biol. 2010;26:445–69.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  5. Xing L, Zhao Y, Gao J, Xiang C, Zhu JK. The ABA receptor PYL9 together with PYL8 plays an important role in regulating lateral root growth. Sci Rep. 2016;6:27177.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. Weng JK, Ye M, Li B, Noel JP. Co-evolution of hormone metabolism and signalling networks expands plant adaptive plasticity. Cell. 2016;166(4):881–93.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  7. Lee SC, Luan S. ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ. 2012;35(1):53–60.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. Zhu JK. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol. 2002;53:247–73.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. Zhao Y, Gao J, Im KJ, Chen K, Bressan RA, Zhu JK. Control of plant water use by ABA induction of senescence and dormancy: an overlooked lesson from evolution. Plant Cell Physiol. 2017;58(8):1319–27.

    CAS  PubMed  Article  Google Scholar 

  10. Umezawa T, Nakashima K, Miyakawa T, Kuromori T, Tanokura M, Shinozaki K, Yamaguchi-Shinozaki K. Molecular basis of the Core regulatory network in ABA responses: sensing, signaling and transport. Plant Cell Physiol. 2010;51(11):1821–39.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Fujii H, Chinnusamy V, Rodrigues A, Rubio S, Antoni R, Park SY, et al. In vitro reconstitution of an abscisic acid signalling pathway. Nature. 2009;462(7273):660–4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Dupeux F, Santiago J, Betz K, Twycross J, Park SY, Rodriguez L, et al. A thermodynamic switch modulates abscisic acid receptor sensitivity. EMBO J. 2011;30(20):4171–84.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Park SY, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, et al. Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins. Science. 2009;324(5930):1068–71.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Ma Y, Szostkiewicz I, Korte A, Moes D, Yang Y, Christmann A, et al. Regulators of PP2C phosphatase activity functions as abscisic acid sensors. Science. 2009;324(5930):1064–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Santiago J, Rodrigues A, Saez A, Rubio S, Antoni R, Dupeux F, et al. Modulation of drought resistance by the abscisic acid receptor PYL5 through inhibition of clade a PP2Cs. Plant J. 2009;60(4):575–88.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  16. Fujii H, Verslues PE, Zhu JK. Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. Plant Cell. 2007;19(2):485–94.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Johnson RR, Wagner RL, Verhey SD, Walker-Simmons MK. The abscisic acid-responsive kinase PKABA1 interacts with a seed-specific abscisic acid response element-binding factor, TaABF, and phosphorylates TaABF peptide sequences. Plant Physiol. 2002;130(2):837–46.

    PubMed  PubMed Central  Article  Google Scholar 

  18. Kobayashi Y, Murata M, Minami H, Yamamoto S, Kagaya Y, Hobo T, et al. Abscisic acid-activated SNRK2 protein kinases function in the gene-regulation pathway of ABA signal transduction by phosphorylating ABA response element-binding factors. Plant J. 2005;44(6):939–49.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. Furihata T, Maruyama K, Fujita Y, Umezawa T, Yoshida R, Shinozaki K, et al. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1. Proc Natl Acad Sci U S A. 2006;103(6):1988–93.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. Hao Q, Yin P, Li W, Wang L, Yan C, Lin Z, et al. The molecular basis of ABA independent inhibition of PP2Cs by a subclass of PYL proteins. Mol Cell. 2011;42(5):662–72.

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  21. Melcher K, Ng LM, Zhou XE, Soon FF, Xu Y, Suino-Powell KM, et al. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors. Nature. 2009;462(7273):602–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Santiago J, Dupeux F, Round A, Antoni R, Park SY, Jamin M, et al. The abscisic acid receptor PYR1 in complex with abscisic acid. Nature. 2009;462(7273):665–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  23. Yin P, Fan H, Hao Q, Yuan X, Wu D, Pang Y, et al. Structural insights into the mechanism of abscisic acid signalling by PYL proteins. Nat Struct Mol Biol. 2009;16(12):1230–7.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. Nishimura N, Sarkeshik A, Nito K, Park SY, Wang A, Carvalho PC, et al. PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. Plant J. 2010;61(2):290–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. Li J, Shi C, Sun D, He Y, Lai C, Lv P, et al. The HAB1 PP2C is inhibited by ABA-dependent PYL10 interaction. Sci Rep. 2015;5:10890.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Kim N, Moon SJ, Min MK, Choi EH, Kim JA, Koh EY, et al. Functional characterization and reconstitution of ABA signalling components using transient gene expression in rice protoplasts. Front Plant Sci. 2015;6:614.

    PubMed  PubMed Central  Google Scholar 

  27. Santiago J, Dupeux F, Betz K, Antoni R, Gonzalez-Guzman M, Rodriguez L, et al. Structural insights into PYR/PYL/RCAR ABA receptors and PP2Cs. Plant Sci. 2012;182:3–11.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. Zhao Y, Xing L, Wang X, Hou YJ, Gao J, Wang P, et al. The ABA receptor PYL8 promotes lateral root growth by enhancing MYB77 dependent transcription of auxin-responsive genes. Sci Signal. 2014;7(328):ra53.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  29. Gonzalez-Guzman M, Pizzio GA, Antoni R, Vera-Sirera F, Merilo E, Bassel GW, et al. Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid. Plant Cell. 2012;24(6):2483–96.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. Fuchs S, Tischer SV, Wunschel C, Christmann A, Grill E. Abscisic acid sensor RCAR7/PYL13, specific regulator of protein phosphatase coreceptors. Proc Natl Acad Sci U S A. 2014;111(15):5741–6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Antoni R, Gonzalez-Guzman M, Rodriguez L, Peirats-Llobet M, Pizzio GA, Fernandez MA, et al. Pyrabactin resistance1-like8 plays an important role for the regulation of abscisic acid signalling in root. Plant Physiol. 2013;161(2):931–41.

    CAS  PubMed  Article  Google Scholar 

  32. Zhao Y, Chan Z, Gao J, Xing L, Cao M, Yu C, et al. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proc Natl Acad Sci U S A. 2016;113(7):1949–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Szostkiewicz I, Richter K, Kepka M, Demmel S, Ma Y, Korte A, et al. Closely related receptor complexes differ in their ABA selectivity and sensitivity. Plant J. 2010;61(1):25–35.

    CAS  PubMed  Article  Google Scholar 

  34. Tischer SV, Wunschel C, Papacek M, Kleigrewe K, Hofmann T, Christmann A, et al. Combinatorial interaction network of abscisic acid receptors and coreceptors from Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2017;114(38):10280–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. Yizhou W, Zhong-Hua C, Ben Z, Adrian H, Blatt MR. PYR/PYL/RCAR abscisic acid receptors regulate K+ and cl− channels through reactive oxygen species-mediated activation of Ca2+ channels at the plasma membrane of intact Arabidopsis guard cells. Plant Physiol. 2013;163(2):566–77.

    Article  CAS  Google Scholar 

  36. Zhao J, Zhao L, Zhang M, Zafar SA, Fang J, Li M, et al. Arabidopsis E3 ubiquitin ligases PUB22 and PUB23 negatively regulate drought tolerance by targeting ABA receptor PYL9 for degradation. Int J Mol Sci. 2017;18(9):E1841.

    PubMed  Article  CAS  Google Scholar 

  37. Park SY, Peterson FC, Mosquna A, Yao J, Volkman BF, Cutler SR. Agrochemical control of plant water use using engineered abscisic acid receptors. Nature. 2015;520(7548):545–8.

    PubMed  Article  CAS  Google Scholar 

  38. Zhang F, Lu X, Lv Z, Zhang L, Zhu M, Jiang W, et al. Overexpression of the Artemisia orthologue of ABA receptor, AaPYL9, enhances ABA sensitivity and improves artemisinin content in Artemisia annua L. PLoS One. 2013;8(2):e56697.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Boneh U, Biton I, Zheng C, Schwartz A, Ben-Ari G. Characterization of potential ABA receptors in Vitis vinifera. Plant Cell Rep. 2012;31(2):311–21.

    CAS  PubMed  Article  Google Scholar 

  40. Gao Z, Li Q, Li J, Chen Y, Luo M, Li H, et al. Characterization of the ABA receptor VlPYL1 that regulates anthocyanin accumulation in grape berry skin. Front Plant Sci. 2018;9:592.

    PubMed  PubMed Central  Article  Google Scholar 

  41. Kim H, Hwang H, Hong J, Lee Y, Ahn IP, Yoon IS, et al. A rice orthologue of the ABA receptor, OsPYL/RCAR5, is a positive regulator of the ABA signal transduction pathway in seed germination and early seedling growth. J Exp Bot. 2012;63(2):1013–24.

    CAS  PubMed  Article  Google Scholar 

  42. Tian X, Wang Z, Li X, Lv T, Liu H, Wang L, et al. Characterization and functional analysis of Pyrabactin resistance-like abscisic acid receptor family in rice. Rice (N Y). 2015;8(1):28.

    Article  Google Scholar 

  43. He Y, Hao Q, Li W, Yan C, Yan N, Yin P. Identification and characterization of ABA receptors in Oryza sativa. PLoS One. 2014;9(4):e95246.

    PubMed  PubMed Central  Article  Google Scholar 

  44. Kim H, Lee K, Hwang H, Bhatnagar N, Kim DY, Yoon IS, et al. Overexpression of PYL5 in rice enhances drought tolerance, inhibits growth, and modulates gene expression. J Exp Bot. 2014;65(2):453–64.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. Miao C, Xiao L, Hua K, Zou C, Zhao Y, Bressan RA, et al. Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity. Proc Natl Acad Sci U S A. 2018;115(23):6058–63.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  46. Chen ZQ, Kong L, Zhou Y, Chen ZJ, Tian DG, Lin Y, et al. Endosperm-specific OsPYL8 and OsPYL9 act as positive regulators of the ABA signaling pathway in rice seed germination. Funct Plant Biol. 2017;44(6):635–45.

    CAS  PubMed  Article  Google Scholar 

  47. Verma RK, Santosh Kumar VV, Yadav SK, Pushkar S, Rao MV. Chinnusamy V overexpression of ABA receptor PYL10 gene confers drought and cold tolerance to indica rice. Front Plant Sci. 2019;10:1488.

    PubMed  PubMed Central  Article  Google Scholar 

  48. Gordon CS, Rajagopalan N, Risseeuw EP, Surpin M, Ball FJ, Barber CJ, et al. Characterization of Triticum aestivum abscisic acid receptors and a possible role for these in mediating Fusairum head blight susceptibility in wheat. PLoS One. 2016;11(10):e0164996.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. Mega R, Abe F, Kim SK, Tsuboi Y, Tanaka K, Kobayashi H, et al. Tuning water-use efficiency and drought tolerance in wheat using abscisic acid receptors. Nat Plants. 2019;5(2):153–9.

    CAS  PubMed  Article  Google Scholar 

  50. Wang YG, Fu FL, Yu HQ, Hu T, Zhang YY, Tao Y, et al. Interaction network of core ABA signalling components in maize. Plant Mol Biol. 2018;96(3):245–63.

    CAS  PubMed  Article  Google Scholar 

  51. Fan W, Zhao M, Li S, Xue B, Jia L, Meng H, et al. Contrasting transcriptional responses of PYR1/PYL/RCAR ABA receptors to ABA or dehydration stress between maize seedling leaves and roots. BMC Plant Biol. 2016;16(1):99.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  52. Gonzãlez-Guzmãn M, Rodrìguez L, Lorenzo-Orts L, Pons C, Sarrión Perdigones A, Fernãndez MA, et al. Tomato PYR/PYL/RCAR abscisic acid receptors show high expression in root, differential sensitivity to the abscisic acid agonist quinabactin, and the capability to enhance plant drought resistance. J Exp Bot. 2014;65(15):4451–64.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  53. Bai G, Yang D-H, Zhao Y, Ha S, Yang F, Ma J, et al. Interactions between soybean ABA receptors and type 2C protein phosphatases. Plant Mol Biol. 2013;83(6):651–64.

    CAS  PubMed  Article  Google Scholar 

  54. Yu J, Yang L, Liu X, Tang R, Wang Y, Ge H, et al. Overexpression of poplar Pyrabactin resistance-like abscisic acid receptors promotes abscisic acid sensitivity and drought resistance in transgenic Arabidopsis. PLoS One. 2016;11(12):e0168040.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  55. Guo D, Zhou Y, Li HL, Zhu JH, Wang Y, Chen XT, et al. Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree. Sci Rep. 2017;7:45157.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. Chai YM, Jia HF, Li CL, Dong QH, Shen YY. FaPYR1 is involved in strawberry fruit ripening. J Exp Bot. 2011;62(14):5079–89.

    CAS  PubMed  Article  Google Scholar 

  57. Chen Y, Feng L, Wei N, Liu ZH, Hu S, Li XB. Overexpression of cotton PYL genes in Arabidopsis enhances the transgenic plant tolerance to drought stress. Plant Physiol Biochem. 2017;115:229–38.

    CAS  PubMed  Article  Google Scholar 

  58. Liang C, Liu Y, Li Y, Meng Z, Yan R, Zhu T, et al. Activation of ABA receptors gene GhPYL9-11A is positively correlated with cotton drought tolerance in transgenic Arabidopsis. Front Plant Sci. 2017;8:1453.

    PubMed  PubMed Central  Article  Google Scholar 

  59. Zhang G, Lu T, Miao W, Sun L, Tian M, Wang J, et al. Genome-wide identification of ABA receptor PYL family and expression analysis of PYLs in response to ABA and osmotic stress in Gossypium. PeerJ. 2017;5:e4126.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  60. Li Y, Wang D, Sun C, Hu X, Mu X, Hu J, et al. Molecular characterization of an AtPYL1-like protein, BrPYL1, as a putative ABA receptor in Brassica rapa. Biochem Biophys Res Commun. 2017;487(3):684–9.

    CAS  PubMed  Article  Google Scholar 

  61. Pri-Tal O, Shaar-Moshe L, Wiseglass G, Peleg Z, Mosquna A. Non-redundant functions of the dimeric ABA receptor BdPYL1 in the grass Brachypodium. Plant J. 2017;92(5):774–86.

    CAS  PubMed  Article  Google Scholar 

  62. Zhang F, Wei Q, Shi J, Jin X, He Y, Zhang Y, et al. Brachypodium distachyon BdPP2CA6 interacts with BdPYLs and BdSnRK2 and positively regulates salt tolerance in transgenic Arabidopsis. Front Plant Sci. 2017;8:264.

    PubMed  PubMed Central  Google Scholar 

  63. Hanada K, Hase T, Toyoda T, Shinozaki K, Okamoto M. Origin and evolution of genes related to ABA metabolism and its signalling pathways. J Plant Res. 2011;124(4):455–65.

    CAS  PubMed  Article  Google Scholar 

  64. Li C, Jia H, Chai Y, Shen Y. Abscisic acid perception and signalling transduction in strawberry: a model for non-climacteric fruit ripening. Plant Signal Behav. 2011;6(12):1950–3.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Wang Y, Wu Y, Duan C, Chen P, Li Q, Dai S, et al. The expression profiling of the CsPYL, CsPP2C and CsSnRK2 gene families during fruit development and drought stress in cucumber. J Plant Physiol. 2012;169(18):1874–82.

    CAS  PubMed  Article  Google Scholar 

  66. Pereira A. Plant abiotic stress challenges from the changing environment. Front Plant Sci. 2016;7:1123.

    PubMed  PubMed Central  Google Scholar 

  67. Sakai H, Lee SS, Tanaka T, Numa H, Kim J, Kawahara Y, et al. Rice annotation project database (RAP-DB): an integrative and interactive database for rice genomics. Plant Cell Physiol. 2013;54(2):e6.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. Kawahara Y, de la Bastide M, Hamilton JP, Kanamori H, McCombie WR, Ouyang S, et al. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data. Rice (N Y). 2013;6(1):4.

    Article  Google Scholar 

  69. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol. 2003;52(5):696–704.

    PubMed  Article  Google Scholar 

  70. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31(8):1296–7.

    PubMed  Article  PubMed Central  Google Scholar 

  72. Cannon SB, Mitra A, Baumgarten A, Young ND, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biol. 2004;4:10.

    PubMed  PubMed Central  Article  Google Scholar 

  73. Dai X, Zhao PX. psRNATarget: a plant small RNA target analysis server. Nucleic Acids Res. 2018;46(W1):W49–54.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  74. Kozomara A, Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 2014;42(Database issue):D68–73.

    CAS  PubMed  Article  Google Scholar 

  75. Kaur V, Yadav SK, Wankhede DP, Pulivendula P, Kumar A, Chinnusamy V. Cloning and characterization of a gene encoding MIZ1, a domain of unknown function protein and its role in salt and drought stress in rice. Protoplasma. 2020;257(2):475–87.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  76. Mansueto L, Fuentes RR, Borja FN, Detras J, Abriol-Santos JM, Chebotarov D, et al. Rice SNP-seek database update: new SNPs, indels, and queries. Nucleic Acids Res. 2017;45(D1):D1075–81.

    CAS  PubMed  Article  Google Scholar 

  77. Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Res. 1999;27(1):297–300.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  78. Mena M, Vicente-Carbajosa J, Schmidt RJ, Carbonero P. An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm. Plant J. 1998;16(1):53–62.

    CAS  PubMed  Article  Google Scholar 

  79. Stalberg K, Ellerstom M, Ezcurra I, Ablov S, Rask L. Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta. 1996;199(4):515–9.

    CAS  PubMed  Article  Google Scholar 

  80. Suzuki M, Ketterling MG, McCarty DR. Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1- and CBF/DREB1-regulated genes of Arabidopsis. Plant Physiol. 2005;139(1):437–47.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  81. Plesch G, Ehrhardt T, Mueller-Roeber B. Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-specific gene expression. Plant J. 2001;28(4):455–64.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  82. Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, et al. Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinforma. 2008;2008:420747.

    Google Scholar 

  83. Dalal M, Inupakutika M. Transcriptional regulation of ABA core signalling component genes in sorghum (Sorghum bicolor L. Moench). Mol Breed. 2014;34:1517–25.

    CAS  Article  Google Scholar 

  84. Gallegos JE, Rose AB. An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana. Sci Rep. 2019;9(1):13777.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  85. Di F, Jian H, Wang T, Chen X, Ding Y, Du H, et al. Genome-wide analysis of the PYL gene family and identification of PYL genes that respond to abiotic stress in Brassica napus. Genes. 2018;9(3):156.

    PubMed Central  Article  CAS  Google Scholar 

  86. Yan J, Wang P, Wang B, Hsu C-C, Tang K, Zhang H, et al. The SnRK2 kinases modulate miRNA accumulation in Arabidopsis. PLoS Genet. 2017;13(4):e1006753.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  87. Lenka SK, Muthusamy SK, Chinnusamy V, Bansal KC. Ectopic expression of rice PYL3 enhances cold and drought tolerance in Arabidopsis thaliana. Mol Biotechnol. 2018;60(5):350–61.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  88. Haralampidis K, Milioni D, Rigas S, Hatzopoulos P. Combinatorial interaction of cis elements specifies the expression of the Arabidopsis AtHsp90-1 gene. Plant Physiol. 2002;129(3):1138–49.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  89. Berardini TZ, Reiser L, Li D, Mezheritsky Y, Muller R, Strait E, et al. The Arabidopsis information resource: making and mining the “gold standard” annotated reference plant genome. Genesis. 2015;53(8):474–85.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  91. Bailey TL, Elkan C. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol. 1994;2:28–36.

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(Web Server issue):W202–8.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  93. Zimmermann L, Stephens A, Nam SZ, Rau D, Kübler J, Lozajic M, et al. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J Mol Biol. 2018;430(15):2237–43.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  94. Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13(11):2498–504.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  95. Gurjar AKS, Panwar AS, Gupta R, Mantri SS. PmiRExAt: plant miRNA expression atlas database and web applications. Database (Oxford). 2016;2016:baw060.

    Article  CAS  Google Scholar 

  96. Wang Y, Tang H, DeBarry JD, Tan X, Li J, Wang X, et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012;40(7):e49.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  97. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, Jones SJ, Marra MA. Circos: an information aesthetic for comparative genomics. Genome Res. 2009;19(9):1639–45.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  98. Madeira F, Park YM, Lee J, Buso N, Gur T, Madhusoodanan N, Basutkar P, Tivey ARN, Potter SC, Finn RD. Lopez R; the EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Res. 2019;47(W1):W636–41.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  99. Suyama M, Torrents D, Bork P. PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Res. 2006;34(Web Server issue):W609–12.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  100. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402–8.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  101. Jain M, Nijhawan A, Tyagi AK, Khurana JP. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun. 2006;345(2):646–51.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank Indian Council of Agricultural Research (ICAR)-Indian Agricultural Research Institute, New Delhi for providing all the facilities for the research work.

Funding

This work was funded by ICAR-National agricultural science fund (NASF) grant no. NFBSFARA/Phen-2015, NAHEP, Indian Council of Agricultural Research (ICAR), New Delhi, grant No. NAHEP/CAAST/2018–19/07 and IARI in-house project grant no. CRSCIARISIL20144047279.

Author information

Affiliations

  1. Division of Plant Physiology, ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi, 110012, India

    Shashank Kumar Yadav, Vinjamuri Venkata Santosh Kumar, Rakesh Kumar Verma, Pragya Yadav & Viswanathan Chinnusamy

  2. School of Biotechnology, Gautam Buddha University, Greater Noida, UP, 201310, India

    Shashank Kumar Yadav & Bhupendra Chaudhary

  3. ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi, 110012, India

    Ankit Saroha & Dhammaprakash Pandhari Wankhede

Authors
  1. Shashank Kumar Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vinjamuri Venkata Santosh Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rakesh Kumar Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pragya Yadav
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ankit Saroha
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dhammaprakash Pandhari Wankhede
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bhupendra Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Viswanathan Chinnusamy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization of research (SKY, BC and VC); Methodology (SKY, VVSK, RKV and PY); q-RT-PCR expression analysis (SKY, VVSK and RKV); In silico promoter analysis and miRNA identification (PY); Collinearity and synteny analysis (SKY, AS and DPW); Data Analysis (All authors); Manuscript writing (SKY, BC and VC); Supervision (VC). All authors read and approved the final manuscript.

Corresponding author

Correspondence to Viswanathan Chinnusamy.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1

Complete CDS sequences of 13Nagina22 OsPYLs.

Additional file 2.

Sequence of 13 Nagina22 OsPYL proteins.

Additional file 3 : Table S1.

Putative function of Motifs identified.

Additional file 4.

Protein sequence of identified PYLs of eight species.

Additional file 5 : Table S2.

Pair wise collinearity among PYLs of Arabidopsis and rice.

Additional file 6 : Figure S1.

Synteny blocks between Arabidopsis and rice at genomic level.

Additional file 7.

Ka/Ks values of PYL orthologs.

Additional file 8

Sequence and detail of identified miRNAs targeting OsPYLs.

Additional file 9 : Figure S2.

Sequence alignment of 13 OsPYL proteins depicting four conserved loops CL1–CL4.

Additional file 10

List of identified CRE and their putative function in 13 OsPYL promoter.

Additional file 11 : Figure S3.

Frequency and distribution of identified CRE in individual OsPYL promoter.

Additional file 12 : Table S3.

List of homologous gene pair between Arabidopsis and rice.

Additional file 13 : Table S4.

List of primers used for q-RT expression analysis of 13 OsPYL genes.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yadav, S.K., Santosh Kumar, V.V., Verma, R.K. et al. Genome-wide identification and characterization of ABA receptor PYL gene family in rice. BMC Genomics 21, 676 (2020). https://doi.org/10.1186/s12864-020-07083-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12864-020-07083-y

Keywords

  • ABA receptors (ABARs)
  • Abiotic stresses
  • Collinearity
  • miRNAs
  • Single amino acid polymorphism (SAP)
  • Single nucleotide polymorphism (SNP)
  • Stress responsive cis-elements
  • Synteny

BMC Genomics

ISSN: 1471-2164

Contact us