CBL-interacting protein kinases (CIPKs) in Chickpea: Genome-wide identification, structure and expression analysis under abiotic stresses and development

Calcineurin B-like proteins (CBL)-interacting protein kinases (CIPKs) by interacting with CBLs regulate developmental processes, hormone signalling transduction and mediate stress responses in plants. Although the genome of chickpea is available, information of CIPK gene family has been missing in chickpea. Here, a total of 22 CIPK encoding genes were identified in chickpea and characterized by in silico methods. We found a high structural conservation in chickpea CIPK family. Our analysis showed that chickpea CIPKs have evolved with dicots from common ancestors, and extensive gene duplication events have played an important role in evolution and expansion of CIPK family in chickpea. Most chickpea CIPK proteins localize in cytoplasm and nucleus. Promoter analysis revealed various cis-regulatory elements related to plant development, hormone signaling and abiotic stresses. Expression analysis indicated that CIPKs are significantly expressed in a spectrum of developmental stages, tissue/organs that hinted their important role in plant development. Several CIPK genes had specific and overlapping expressions in different abiotic stresses and seed development stages, suggesting the important role of CIPK family in abiotic stress signaling, and seed development in chickpea. Thus, this study provides the avenue for detailed functional characterization of CIPK family in chickpea and other legume crops.


Introduction
South Asia is the major producer of the world's second most important food legume chickpea. Importantly, India is the largest producer of chickpeas and is credited for about 70% of world's chickpeas production. India contributes an estimated production of 5.9 million tonnes (mt) annually [1]. Chickpea is an important dietary source for vegetarians due to vital nutritive constituents in its seeds, including 20-30% crude protein, 40% carbohydrate, and 3-6% oil [2]. In addition, chickpea seeds are rich in minerals, such as calcium, magnesium, potassium, phosphorus, iron and zinc [3]. The chickpea production is severely affected by various stresses. Consequently, a huge gap is developed between its demand and supply. Abiotic stresses alone account for an estimated 40-60% global chickpea production losses annually. Drought causes major damage and accounts for about 50% of chickpea yield loss. Temperature fluctuations and soil salinity combined are responsible for about 25% of chickpea yield loss [4]. The chickpea yield loss due to drought, cold and salinity, respectively costed approximately 1.3 billion, 186 million and 354 million US dollars, which have economically dented several chickpea-producing countries [5]. These stresses have an adverse effect on flower set, pollen viability, pod set/abortion and retention. As all these crucial developmental stages essentially determine seed number, a negative impact on these stages significantly hampers chickpea yield. Due to continuous climate change, severe and frequent challenges of drought in arid and semiarid areas where chickpea is traditionally cultivated are predicted [6] and that can be detrimental for overall productivity of chickpea. Thus, identification and utilization of important stress related genes in biotechnological programmes to generate improved chickpea varieties is need of the hour.
Environmental cues, such as biotic and abiotic stresses are known to elicit the increase in cytosolic Ca2+ with specific spatio-temporal features. The spatio-temporal accumulation of Ca2+ generates specific "Ca2+ signature" in the form of spikes, waves and oscillations. The stimulus specific Ca2+ signature is decoded by Ca2+ sensors and downstream effectors towards a response [7]. Several Ca2+ sensors have been identified and characterized in plants, including calmodulin (CaM) and CaM-like proteins (CMLs) [8], Ca2+ -dependent protein kinases (CDPKs) [9], and calcineurin B-like proteins (CBLs) [1]. Among these, CBLs are a unique group of Ca2+ sensors and to determine their functional identity, a family of plant specific serine/threonine kinases; CBL-interacting protein kinases (CIPKs) functions as important downstream signaling component [10]. Both CBLs and CIPKs are encoded as multi-gene families in higher plants, for example: 10 CBL and 26 CIPKs members have been identified in Arabidopsis, and 10 CBL and 30 CIPKs in rice [11]. Large numbers of CBL and CIPK members in higher plants constitute a complex and sophisticated signaling network. For instance, In Arabidopsis, each CBL interacts with multiple CIPKs and vice-versa [12], consequently, some CBLs share a common CIPK partner and some CIPKs are regulated by a common CBL. Such specific and overlapping patterns of CBL-CIPK interactions may provide functional specificity and synergism to CBL-CIPK signalling networks.
Structurally, CBLs are typical Ca2+ sensor proteins with four EF-hand domains which are responsible for Ca2+ binding. On the other hand, CIPKs harbours several functionally distinct domains. All CIPKs consist of a conserved catalytic kinase domain at the N-terminal and a regulatory domain at the C-terminal [10,12]. A typical of a functional kinase protein, CIPK kinase domain contains an ATP binding site and an activation loop. The regulatory domain contains FISL/NAF and PPI motifs which are responsible for the interaction with CBL and type 2C protein phosphatases, respectively [13,14]. The function of CBL-CIPK pathways could be regulated by pattern of gene expression, Ca2+ binding affinity, protein stability and protein-protein interactions [7]. CBL-CIPK networks have been implicated in diverse functions that regulate plant response to biotic stress [15-17], abiotic stress [1,18,19], nutrient deficiency [1,20,21] metal toxicity [22,23] and plant development [24][25][26][27]. Majority of the knowledge about CBL-CIPK signaling has developed from the research with model plant Arabidopsis thaliana. Information about CBL-CIPK networks and their role is scarce in important legume crop chickpea. Though, CBL family has been identified in chickpea [28], identification and characterization of CIPK family is missing. A comprehensive gene expression profiling of the CIPK family will help in understanding CBL-CIPK functions in chickpea. Information obtained from expression analysis will encourage the utilization of crucial genes for genetically engineering the chickpea plant towards better stress tolerance and development.
With this rationale, we have identified the CIPK gene family in chickpea. Phylogenetic analysis and chromosomal localization have provided insight into the evolution and expansion of chickpea CIPK family. Analysis of gene and domain structure ensured the authenticity and integrity of identified genes. Homology modelling helped to understand the three-dimensional structure of chickpea CIPKs. In-silico analysis revealed various stress, hormone and development related cis-regulatory elements in CIPK promoters. Expression profiling using various datasets in public repositories suggested involvement of the CIPK family in biotic and abiotic stress signaling, and seed development in chickpea.

Identification of CIPKs in the chickpea genome
The chickpea genome submitted by Varshney et al. (2013) was downloaded from the NCBI and explored to identify CIPK encoding genes. Rice and Arabidopsis thaliana CIPK proteins were retrieved from Uniprot (Swiss-Prot), and homology search was performed using BLAST tool (E-value =10-6) against the chickpea proteome. Significant hits were selected on the basis of >=50% identity, and >=100 amino acid length alignment. Further, the HMM sequence of CIPK-NAF domain was extracted from Pfam (http://pfam.xfam.org/) database, and BLAST search was done (E-value =10) against the chickpea proteome. Furthermore, both the sets of putative candidates were mixed, and redundant sequences were removed using CD-HIT [29]. The domain analysis was performed by using a standalone version of the InterproScan [30]. The gene attributes such as gene ID, protein ID, CDS, size of amino acid and chromosomal coordinates were extracted from NCBI web server.

Phylogenetic analysis
To examine the evolutionary relationship between CIPKs in chickpea and other species, Multiple Sequence Alignment (MSA) was performed with the amino acid sequences of CIPKs from four different plant species e.g., Arabidopsis thaliana, Oryza sativa, Glycine max, and Cicer arietinum) using ClustalW [31] at default settings in MEGA X version 10. 1.8 [32]. The neighbour-joining method was used to construct the phylogenetic tree and bootstrap values were calculated in 1000 replicates to determine the phylogenetic relationship among the CIPKs. iTOL [33] webserver was used to mark the different clades of CIPKs with different colours and shapes for better visualization.

Gene structures, motif organization and domain prediction
Gene Structure Display Server (GSDS) program (http://gsds.gao-lab.org/) was used to compare the CDS sequences with their corresponding genomic DNA sequences in order to investigate the coding sequences and intron structure. Motif organization of CIPK proteins was examined via the Multiple Expression motifs for Motif Elicitation (MEME) tool [34] with default parameters; site distribution -zero or one occurrence per sequence; motif discovery mode -classic; motif length 6-50; and the top ten most enriched motifs were selected based on lowest E-values. The identification of domains was performed by a standalone package of InterProScan [30]. The coordinates of the essential domains and active sites were extracted and used as input in Illustrator for Biological Sequences [35] for the visualization.

Gene nomenclature, chromosomal location and gene duplication
The names of CIPK genes in chickpea were assigned according to their closest orthologous relationship with Arabidopsis CIPK genes in the phylogenetic tree. The information of the chromosome coordinates was obtained from NCBI. Their localization was displayed in different chromosomes using TBtools [36]. To search for all duplicated gene pairs within the chickpea genome, the protein sequence of chickpea was used to run the all-versus-all local BLASTP with parameters of E-value 1e-5, max target sequences 5, and m6 format output. MCScanX software package [37] was used to analyse the segmentally duplicated regions of CIPK genes of chickpea. The genes and the intra-species collinear gene pairs were mapped to the eight chromosomes of chickpea using the family_cir-cle_plotter.java script. The protein sequences of each duplicate gene pair were aligned by CLUSTALW. The alignment file in FASTA format and the CDS sequences of the corresponding genes were used to calculate the non-synonymous (Ka) and synonymous (Ks) substitution values by the PAL2NAL server [38].

In silico promoter analysis
For the identification of cis-regulatory elements in the promoters of chickpea CIPKs, 2000 bp upstream sequences of the coding region of genes were extracted from NCBI and used as input in the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) tool.

Subcellular localization and physicochemical properties of CaCIPK proteins
The full-length protein sequences of all the CIPKs of chickpea were used as input to predict their subcellular localization using the CELLO program [39]. The locations were displayed in different parts of the cell by Biorender software (https://biorender.com/). The online tool Compute pI/MW of Expasy [40] was used to calculate the molecular weight (MW) and isoelectric point (pI) of CaCIPKs.

Protein-Protein Interaction Network Construction for CaCBLs -CaCIPKs
To elucidate the interaction network between CIPK and CBL proteins in chickpea, the amino acid sequences of 22 CIPKs, and 9 CBLs from the study of Meena et al., 2015 were used as input in STRING (http://string-db.org/). At STRING, the interaction network can be constructed using low confidence value of 0.15, medium confidence of 0.4, high confidence of 0.7 and highest confidence of 0.9. Experimental data of interacting CBL and CIPK proteins in Arabidopsis were constructed using the confidence value > 0.4. Homologous proteins of the determined interactive Arabidopsis proteins in chickpea were identified by reciprocal best BLASTP analysis.

Protein tertiary structure prediction
The tertiary structures of the 22 CaCIPK proteins were predicted with the Phyre2 web portal (http://www.sbg.bio.ic.ac.uk/phyre2). Phyre2 uses advanced remote homology detection methods to build 3D models for protein sequences [41]. All the proteins were modelled with 100% confidence by the single highest scoring template model.

Expression analysis using RNA-seq data
To generate the genome-wide expression profiles of CaCIPK genes in different tissues and developmental stages, RNA-Seq data was extracted from NCBI Sequence Read Archive; SRA number SRP121085. RNA-seq data for different seed stages in two distinct desi chickpea varieties (JGK3 and Himchana 1) was extracted from SRA number SRP072563 and SRP072564. Data for three abiotic stresses (desiccation, cold and salinity stress) in root, and shoot tissue of ICC4958 chickpea variety was extracted from SRA number SRP034839.
The raw reads downloaded from SRA at NCBI were processed using FASTP [42] to remove the adapter, poly-N, short and low-quality reads. The reference genome of chickpea was downloaded from the NCBI genome web server. The HISAT2 [43] tool was used for building the index of the reference genome, and for mapping of filtered reads onto the genome. The alignments were assembled into potential transcripts using StringTie [44] and the transcript abundance was calculated as fragments per kilobase of transcript per million reads (FPKM) values. For differential expression analysis, three biological replicates of each treatment and control were analysed and fold change expression was calculated by the ratio of average FPKM of test samples and average FPKM of control samples. The 'pheatmap' package of R was used to generate the heatmaps of the expression data using the logarithm of normalized expression values for the tissue study and the logarithm of fold change for the remaining studies.

Identification and sequence analysis of CaCIPK genes
A total of 39 putative CIPK sequences were obtained from homology search with Arabidopsis and rice CIPKs. Further HMM profile search against chickpea proteome revealed 38 putative CIPK sequences. After combining both sets, followed by manual curation a total of 26 unique CIPK sequences were obtained. Domain analysis revealed that the necessary domains (e.g. PPI and NAF domain) were absent in four sequences, therefore, they were removed from the list. Finally, a total of 22 non-redundant CIPK encoding genes were found in the chickpea genome. Previously, 26 CIPK members have been reported in Arabidopsis The length of 22 CaCIPK proteins varied from 418aa (CaCIPK16) to 503aa (CaCIPK12) with average molecular weight of 51.16 kDa. Most of the CaCIPK proteins (except CaCIPK3 and CaCIPK11) were found to have an isoelectric point (pI) greater than 7 (Table 1).  Figure S1). Even the most divergent protein pair CaCIPK8 and CaCIPK22 shared 35.49% identity (49.18% similarity). These findings suggest that plant CIPKs are highly conserved in terms of sequence and structure, which hints towards their similar mode of action.

Gene and domain structure
The evolution of gene families is often reflected by their gene structure [52, 53]. A large variation in the number of introns was found in CIPK genes in chickpeas with number of introns ranging from 0 to 14 ( Figure 1A). Out of 22 CaCIPK genes, only seven had more than two introns. Thus, CaCIPKs could be classified into two groups: i) intron-poor subgroup with zero (CaCIPK2, -4, -5, -6, -7, -10, -11, -12, -13, -15, -16, -18, -20, -22) or one (CaCIPK14) intron, and ii) intron-rich subgroup with greater than 10 introns (CaCIPK1, -3, -8, -9, -17, -19, -21). Division of CIPKs into two subgroups was also supported by the division of clade in the phylogenetic tree. Similar pattern of intron-rich and intron-poor CIPK genes has been reported in different plant species, including Arabidopsis [54], rice [55], soybean [56], wild sugarcane [57] and wheat [47]. These findings suggest that CIPK gene family is structurally conserved across plant kingdom. The domain structure analysis revealed that all the CaCIPK proteins possess three essential domains, at N-terminal a kinase domain, and at C-terminal regulatory NAF and protein phosphatase interaction (PPI) domain (Figure 1(B)). Kinase domains contain an ATP binding site and an activation loop. During CIPK activity, the stabilization of substrates at the active site is regulated by the phosphorylation of activation loop [58]. The activation loop has been found between the conserved 'DFG' and 'APE' amino acid residues. However, in our analysis, few variations were observed in the conserved short motifs. Alignment of 22 CaCIPK proteins showed that the glycine residue of 'DFG' was changed to asparagine (DFN) in CaCIPK1, whereas the alanine of 'APE' was modified to serine (SPE) in CaCIPK6 and CaCIPK18 ( Figure S2). Importantly, sites important for phosphoregulation of activation loop i.e., serine, threonine, and tyrosine [59] were found to be conserved in all CaCIPK proteins except CaCIPK6 where serine was modified to cysteine. Further investigations are required to assess an effect of these changes on the function of the activation loop. In CIPKs, NAF motif mediates the interaction between CIPK and CBL, and FISL motif keeps the kinase inactive under normal conditions, hence act as autoinhibitory domain [13,14]. PPI domain is required for interaction of CIPKs with protein phosphatase 2C (PP2C) [60]. In CaCIPK proteins, a total of 10 conserved motifs were identified by using the MEME tool. Out of those, motif 7 was annotated as NAF domain due to the presence of conserved asparagine-alanine-phenylalanine residues [13], whereas motif 8 which is located just after the motif 7 was designated as the PPI domain as it contains important arginine and phenylalanine residues. All motifs except motif 6 and 10 were present in 22 CaCIPK proteins ( Figure S3). Motif 6 was absent in CaCIPK4, whereas motif 10 was absent in the subgroup of intron-rich CaCIPKs. This may explain the presence of CaCIPK4 in a separate phylogenetic clade. The similar motif composition of proteins of the same clade was also reported in tomato and saccharum [48,57]. Furthermore, the sequential arrangement and the size of motifs in all the CaCIPK proteins were similar, which hints towards structural conservation and a close phylogenetic relationship, as previously reported in Prunus mume [50]. The sequence logo of different motifs is depicted in Table S1.

Phylogenetic analysis of CaCIPK family
A total of 133 CIPK protein sequences from four species: Arabidopsis thaliana (26), Oryza sativa (33), Glycine max (52), and Cicer arietinum (22) were used to construct the phylogenetic tree to explore the evolutionary relationship among the CIPKs. Based on high bootstrap values, the tree was divided into two major groups: group I and II. Two groups were further sub-divided IA, IB, and IIA, IIB (Figure 2). Interestingly, all intron-rich CaCIPKs were clustered in group I, and intron-poor CaCIPKs were clustered in group II. This indicates evolutionary conservation among intron-rich and intron-poor CIPKs in chickpea, and thus CIPKs may have evolved as two groups in chickpea. Similar phylogenetic pattern has been observed for CIPKs in other plants, such as Zea mays [61] and Prunus mume [50]. Remarkably, CaCIPKs were found to be close to CIPKs from Arabidopsis and soybean, but distantly placed from rice CIPKs. This suggests that CIPKs might have evolved separately in dicots and monocots. Group IIB contained most members of CaCIPKs. Group IA includes CaCIPK1, -17, -21, group IB includes CaCIPK3, -8, -9 and -19, Group IIA includes CaCIPK4, -11, -14 and -22 and Group IIB includes CaCIPK2, -5, -6, -7, -10, -12, -13, -15, -16, -18 and -20.

Chromosomal location and gene duplication of CaCIPK genes
All the CaCIPK genes were mapped onto the seven out of eight chromosomes of chickpea. None of them was localized on chromosome 8. All the genes were unevenly  In our study, the ratio of Ka to Ks for CaCIPKs was calculated which ranged from 0.0068 to 0.1675. Thus, Ka/Ks was found to be less than 1 for all segmentally duplicated gene pairs which suggests that all duplicated genes of CaCIPK family had undergone purifying selection on the whole genome duplication (WGD) ( Table S2).

Subcellular localization and structure prediction
In our study, the majority of CaCIPK proteins were found to localize in the cytoplasm, and only six CaCIPK proteins were localized in the nucleus ( Figure 5). Among 22 CIPKs, five proteins namely CaCIPK2, -6, -10, -13, -19 were found to be localized both in the nucleus and cytosol. The three-dimensional structures of 22 CaCIPK proteins were modelled with 100% confidence by the single highest scoring template ( Figure 6). Majority part of the models were built based on two templates -c6c9dB (Serine/threonine-protein kinase MARK1) and c5ebzF (inhibitor of nuclear factor kappa-b kinase subunit alpha) on the basis of raw alignment score which takes into account sequence and secondary structure similarity, inserts and deletes. CaCIPK16 showed the maximum coverage (95%) whereas CaCIPK12 showed the least coverage (50%). The identity of the template model c4czuC which belonged to CIPK23 with other CIPKs varied from 52% to 85% Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 15 March 2021 doi:10.20944/preprints202103.0369.v1 (Table S4). All the CaCIPK proteins were found to have comparable numbers of α-helices and β-sheets, ranging from 16 to 21 and 14 to 17. The 3D structure of CIPKs have not been fully explored in other plants, however in Prunus mume 3D structures of PmCIPK proteins were predicted which shared the highest identity with the hypothetical protein c6c9dB [50], similar to CaCIPK proteins.

Expression profile of CIPK genes in different developmental stages
The expression analysis of CaCIPKs was carried out in 27 tissues of chickpea belonging to different stages i.e. germination stage (radicle, plumule, embryo), seedling stage (epicotyl, primary root), vegetative stage (root, petiole, stem, leaf), reproductive stage (nodules, flowers, buds, pods, immature seeds), and senescence stage (yellow leaf, immature seeds, mature seeds, seed coat, and nodules) (Figure 8). to have ubiquitously high expression in all the tissues. Whereas CaCIPK2, -8, -13, -17 and -21 showed low expression in almost all the tissues. This expression pattern indicates that CIPKs might be involved in regulation of a wide array of processes during different stages of plant development in chickpea. (Table S6). Similar expression pattern has been observed for CIPKs in plants such as Arabidopsis, rice and wheat [47, 65] AtCIPK19 has been found to express highly in pollen grains and pollen tubes and analysis of atcipk19 mutant and overexpression in plants revealed that AtCIPK19 is required for pollen tube growth and polarity [24]. AtCIPK6 and its chickpea ortholog have been shown to regulate root development via controlling auxin transport in Arabidopsis [26]. Tomato SlCIPK2 is specifically expressed in floral organs and through interaction with different SlCBLs and transcription factors regulates stamen development and stress tolerance [85]. Also, Manihot esculenta CIPKs; MeCIPK16 and MeCIPK20 were predominantly expressed in flowers [86]. These findings suggest that CIPKs are key regulators of plant development.

Expression profile of CIPK genes in abiotic stress
To investigate the possible involvement of chickpea CIPKs in abiotic stress signaling, their expression pattern was analysed in root and shoot under three major abiotic stresses, desiccation, salinity and cold. Several CIPK genes were found to differentially express both in root and shoot (Figure 9). Seven genes, CaCIPK2, -4, -6 -11, -12, -13, and -18 were found to be commonly upregulated in all three abiotic stresses, whereas five genes CaCIPK3, -5, -9, -16 and -21 were commonly downregulated in root (Table S7). Notably, CaCIPK8 was upregulated in cold

Expression profile in different stages of seed development
Optimum development of seeds leads to their production with sufficient quantity as well as quality, thereby it determines the yield. To understand the role of CIPKs in chickpea seed development, expression profile was generated with mature leaf as control and seven different seed stages, representing early-embryogenesis (S1), mid-embryogenesis (S2), late-embryogenesis (S3), mid-maturation (S4-S5), and late-maturation (S6-S7), in two desi cultivars: JGK3 (large seeded) and Himchana1(small seeded) ( Figure 10). Few CaCIPK genes, including CaCIPK2, -11, -13 were ubiquitously expressed during all the seed stages in both the chickpea varieties however, level of expression varied (Table  S8). CaCIPK2 expressed highly during S5-S7 in JGK3 whereas, during S4 in Himchana1. CaCIPK11 showed high expression during S1-S5 in both the varieties and during S7 in Himchana1. Similarly, CaCIPK13 showed significant expression during S1-S5 however, the level of expression was higher in JGK3 than Himchana1. In contrast, CaCIPK18 and -21 showed significant expression during S1-S5, but expression was higher in Himchana1. Remarkably, CaCIPK6 and -16 were upregulated during S1-S4 but downregulated during S5-S7 in both the varieties. CaCIPK10 was upregulated during S4-S5 in both the varieties but upregulated during S6 only in JGK3. Two CaCIPK members, CaCIPK12 and -17 were significantly downregulated during all seed stages in both JGK3 and Himchana1. These findings suggest the crucial role of CIPKs in chickpea seed development. Some CIPKs might be involved in regulating all the seed development stages in both the varieties, whereas some members might regulate specific seed stages in both varieties or any specific variety. Thus, CIPKs could play an important role in determining the chickpea yield.

Preprints
Very few studies have analysed the role of CIPKs in seed development till date. The role of CIPKs in seed development has been proposed in plant species like rice and Phaseolus vulgaris. Along with CBLs, several rice CIPK genes were differentially expressed during five stages of seed development [65]. In Phaseolus vulgaris, PvCIPK1, -2, -3 and -5 were expressed only in small and mid-size developing seeds but show no expression in large developing seeds [92].

Conclusions
CIPKs have been studied at the genome-wide scale in diverse plant species, however, in-depth study of the CIPK gene family was missing in important legume crop chickpea. Therefore, in this study, genome-wide identification and characterization of the CIPK gene family was carried out in chickpea which unearthed a total of 22 CIPK genes. Gene and protein structure analysis indicated structural conservation among chickpea CIPKs and homology with other plant species. Phylogenetic analysis suggests that chickpea CIPKs have evolved from common dicot ancestors and gene duplication is the major driving force behind their evolution and expansion. Subcellular analysis showed that the CIPK proteins are majorly located in the nucleus and cytoplasm. In-silico interaction analysis revealed various specific and overlapping functional complex formations between CBLs and CIPKs which could be tested functionally in future. Expression analysis during various developmental stages indicated that CIPKs are expressed in a wide range of tissue/organs and could play an important role in their development. Promoter and expression analysis of the CIPK gene family strongly suggest their role in abiotic stress signaling and seed development in chickpea. Thus, this study provides a useful platform for detailed functional characterization of the CIPK family in chickpea and other legume crops.
Supplementary Materials: Table S1: Sequence logo of different motifs identified through MEME; Table S2: List of segmentally duplicated gene pairs; Table S3: List of cis-regulatory elements identified in the promoter regions of CaCIPK genes; Table S4: The confidence, coverage and sequence identities of the homologous relationship of the CaCIPKs; Table S5: Type and strength of interactions between CBL and CIPK proteins in chickpea; Table S6: Log2FPKM values of different tissues belonging to different developmental stages; Table S7: Log2 Fold Change values of CaCIPKs in response to abiotic stress; Table S8: Log2 Fold Change values of CaCIPKs in various stages of seed development; Figure S1: Percentage of identity and similarity between CaCIPKs; Figure S2: Alignment of 22 CaCIPKs for domain identification; Figure S3: Identification of motifs through MEME; Figure S4: Duplication of chickpea CIPK genes performed by MCScanX is shown via Circos plot.
Author Contributions: Conceptualization, A.S. and S.K.; data curation, N.P.; writing-original draft preparation, N.P.; writing-review and editing, A.S. and S.K.; supervision, S.K.; project administration, S.K.; funding acquisition, S.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.