3.1. Identification and sequence analysis of CaCIPK genes
In chickpea, a total of 39 putative CIPK protein sequences were obtained by the 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 curations, a total of 26 unique CIPK sequences were obtained. Domain analysis revealed that the necessary domains like 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 fetched from chickpea genome.
The length of 22 CaCIPK proteins varied from 418 aa (CaCIPK16) to 503 aa (CaCIPK12) with an 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). To gain insights into the homology of the CaCIPK proteins, the sequence identity and similarity were calculated using the SIAS tool (http://imed.med.ucm.es/Tools/sias.html). This analysis showed that the CaCIPKs have 46.13 to 81.87% sequence similarity among themselves. Four protein pairs viz. CaCIPK15/18, CaCIPK5/7, CaCIPK1/17 and CaCIPK2/10 showed a high degree of identity i.e., 76.67%, 76.39%, 76.01% and 73.84%, respectively (Figure S1). Even the most divergent protein pair of CaCIPK8 and CaCIPK22 shared 35.49% identity (49.18% similarity).
Table 1
Summary of various features of the chickpea CIPK family.
Transcript | Protein |
Gene Name | NCBI ID | Chromosome | Start | End | NCBI Identifier | Introns | CDS length | NCBI Identifier | Length (aa) | Isoelectric point (pI) | Protein wt. (kDa) |
CaCIPK1 | LOC101499928 | Ca1 | 25702213 | 25709285 | XM_004488130.3 | 11 | 1347 | XP_004488187.1 | 448 | 8.02 | 50.34 |
CaCIPK2 | LOC101488582 | Ca2 | 27180279 | 27182763 | XM_004490648.3 | 0 | 1368 | XP_004490705.1 | 455 | 9 | 51.9 |
CaCIPK3 | LOC101491417 | Ca2 | 33011673 | 33018054 | XM_004491146.3 | 13 | 1326 | XP_004491203.1 | 441 | 6.59 | 50.44 |
CaCIPK4 | LOC101504371 | Ca4 | 32563304 | 32564889 | XM_004497948.3 | 0 | 1305 | XP_004498005.1 | 434 | 9.12 | 49.29 |
CaCIPK5 | LOC101513526 | Ca6 | 53224442 | 53226557 | XM_012717669.2 | 0 | 1353 | XP_012573123.1 | 450 | 8.26 | 51.83 |
CaCIPK6 | LOC101511702 | Ca7 | 1293373 | 1294939 | NM_001309656.1 | 0 | 1335 | NP_001296585.1 | 444 | 9.11 | 50.34 |
CaCIPK7 | LOC101496895 | Ca1 | 8839486 | 8841422 | XM_004486556.3 | 0 | 1347 | XP_004486613.1 | 448 | 9.14 | 50.77 |
CaCIPK8 | LOC101511605 | Ca2 | 23829223 | 23836498 | XM_027331998.1 | 14 | 1425 | XP_027187799.1 | 474 | 8.03 | 53.78 |
CaCIPK9 | LOC101493574 | Ca1 | 36737555 | 36744930 | XM_027334254.1 | 14 | 1398 | XP_027190055.1 | 465 | 8.93 | 52.14 |
CaCIPK10 | LOC101498077 | Ca5 | 26642943 | 26645602 | XM_004500288.3 | 0 | 1392 | XP_004500345.1 | 463 | 8.76 | 52.43 |
CaCIPK11 | LOC101499591 | Ca5 | 26665837 | 26667956 | XM_012715825.2 | 0 | 1320 | XP_012571279.1 | 439 | 6.49 | 48.88 |
CaCIPK12 | LOC101506657 | Ca1 | 3314537 | 3316964 | XM_012712534.2 | 0 | 1512 | XP_012567988.1 | 503 | 7.14 | 56.66 |
CaCIPK13 | LOC101489428 | Ca5 | 28720785 | 28723545 | XM_004500487.3 | 0 | 1380 | XP_004500544.1 | 459 | 9.04 | 51.78 |
CaCIPK14 | LOC101488926 | Ca2 | 27205251 | 27207496 | XM_004490649.3 | 1 | 1308 | XP_004490706.1 | 435 | 8.67 | 48.82 |
CaCIPK15 | LOC101507945 | Ca6 | 37592424 | 37596217 | XM_004506380.3 | 0 | 1395 | XP_004506437.1 | 464 | 8.82 | 52.36 |
CaCIPK16 | LOC101510050 | Ca4 | 44315144 | 44317032 | XM_004498761.3 | 0 | 1257 | XP_004498818.1 | 418 | 8.94 | 47.54 |
CaCIPK17 | LOC101510187 | Ca7 | 3876530 | 3882455 | XM_012717926.2 | 11 | 1344 | XP_012573380.1 | 446 | 8.43 | 50.8 |
CaCIPK18 | LOC101492060 | Ca3 | 21042046 | 21046759 | XM_004492487.3 | 0 | 1392 | XP_004492544.1 | 463 | 8.41 | 52.63 |
CaCIPK19 | LOC101507330 | Ca7 | 6850555 | 6859747 | XM_012718074.2 | 13 | 1341 | XP_012573528.1 | 446 | 9.2 | 50.84 |
CaCIPK20 | LOC101506991 | Ca1 | 3307254 | 3309153 | XM_012712535.2 | 0 | 1362 | XP_012567989.1 | 453 | 8.96 | 52.14 |
CaCIPK21 | LOC101512343 | Ca7 | 2026868 | 2030503 | XM_012717974.2 | 13 | 1359 | XP_012573428.1 | 452 | 8.17 | 51.07 |
CaCIPK22 | LOC101493164 | Ca3 | 21099833 | 21101565 | XM_004492489.3 | 0 | 1296 | XP_004492546.1 | 431 | 8.89 | 48.89 |
3.2. Gene and domain structure
The evolution of gene families is often reflected by their gene structure 50,51. There is a large variation in the number of introns in CIPK genes in chickpea, which ranges from 0 to 14 (Fig. 1A). Out of 22 CaCIPK genes, only seven have 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).
The domain structure analysis revealed that all the CaCIPK proteins possess three essential domains; a N-terminal kinase domain, and at C-terminal, a regulatory NAF domain and protein phosphatase interaction (PPI) domain (Fig. 1B). Multiple sequence alignment of 22 CaCIPK proteins showed that the glycine residue of ‘DFG’ in the activation loop was changed to asparagine (DFN) in CaCIPK1, whereas the alanine of ‘APE’ was modified to serine (SPE) in CaCIPK6 and CaCIPK18 (Figure S2). Important sites for phosphoregulation of activation loop i.e., serine, threonine, and tyrosine 52, were found to be conserved in all CaCIPK proteins except CaCIPK6, where serine was modified to cysteine. 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 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, while motif 10 was absent in the subgroup of intron-rich CaCIPKs. The sequence logo of different motifs is depicted in Table S2.
3.3. Phylogenetic analysis of CaCIPK family
A total of 133 CIPK protein sequences from four species viz. 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 e.g. group I and II. These two groups were further sub-divided into IA, IB, and IIA, IIB (Fig. 2). 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. Group IIB contained most members of CaCIPKs.
3.4. Chromosomal location and gene duplication of CaCIPK genes
All the 22 CaCIPK genes were mapped onto the seven out of eight chromosomes of chickpea (Fig. 3). Chromosome 1 contains the maximum number of genes viz. CIPK1, -7, -9, -12, -20. Chromosomes 2 and 7 harbour four genes each i.e., CIPK2, -3, -8, -14 and CIPK6, -17, -19, -21 respectively. Chromosome 5 contains three genes (CIPK10, -11, -13), that are located very close to each other. The other chromosomes have only two CIPKs each. It was observed that the CaCIPK family had undergone gene duplication as nine gene pairs showed segmental duplications, including CaCIPK1/17, CaCIPK2/10, CaCIPK2/18, CaCIPK5/7, CaCIPK10/18, CaCIPK11/14, CaCIPK11/22, CaCIPK14/22, and CaCIPK15/18 (Figure S4). In addition to this, four gene pairs (e.g. CaCIPK12/20, CaCIPK2/14, CaCIPK18/22, CaCIPK10/11) showed tandem duplications. A ratio of Ka (non-synonymous)/Ks (synonymous substitution) = 1, signifies neutral selection (drift), Ka/Ks < 1 indicates purifying selection, and Ka/Ks > 1 implies positive selection (adaptive evolution) 53. In our study, the ratio of Ka to Ks for CaCIPKs ranged from 0.0068 to 0.1675 (Table S3).
3.5. Cis-regulatory elements in CaCIPK promoters
Various cis-regulatory elements were found to be unevenly distributed on the promoters of CIPK genes in chickpea (Fig. 4). An oxidative stress-responsive element ERE 54 was present in all CaCIPKs. The W box has a role in both biotic and abiotic stress 55, and was present in CaCIPK1, -2, -3, -5, -6, -10, -12, -20, -21, -22. The MYB was present in all the CaCIPK genes except CaCIPK8. The O2 site involved in zein metabolism and circadian motif 54, was present in only six CaCIPK genes viz. CaCIPK2, -5, -13, -16, -19, -20. Another well-characterized cis-element, ABRE involved in abiotic stress, and ABA responsiveness 56,57, was found in the promoter of 15 CaCIPK genes, including CaCIPK1, -2, -3, -4, -5, -6, -7, -8, -10, -11, -14, -17, -18, -19, -22. Besides, LTR-motif was one of the least common elements, and only four CaCIPK genes (CaCIPK2, -10, -19, -21) contain this motif (Table S4).
3.6. Subcellular localization and structure prediction
In our study, the majority of CaCIPK proteins were found to be localized in the cytoplasm, and only six CaCIPK proteins were localized in the nucleus (Fig. 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 (3D) structures of 22 CaCIPK proteins were modelled with 100% confidence by the single highest scoring template (Fig. 6). A major part of the models was based on the two templates e.g. c6c9dB (Serine/threonine-protein kinase MARK1), and c5ebzF (inhibitor of nuclear factor kappa-b kinase subunit alpha), based on raw alignment score which takes into account the sequence and secondary structure similarity, inserts and deletes. The CaCIPK16 showed the maximum coverage (95%), whereas CaCIPK12 showed the least coverage (50%) in the alignment. The identity of the template model c4czuC (belonged to CIPK23) with other CIPKs varied from 52–85% (Table S5). All the CaCIPK proteins were found to have comparable numbers of α-helices and β-sheets, ranging from 16–21, and 14–17 respectively.
3.7. Interaction patterns between CBL and CIPK proteins in chickpea
CIPKs are generally activated by interaction with CBLs to perform different functions. Thus, it is crucial to determine the interactions and functional complexes of CBLs and CIPKs in chickpea. Therefore, in silico analysis was performed to analyze the CBL and CIPK interactions in chickpea. A combined score of co-expression, experimentally determined interaction, and automated text mining was used to predict the strength of interaction. A score of less than 0.7 was taken as weak, whereas a score greater than 0.7 was considered as strong interaction. The CaCBL1 showed strong interaction (thicker lines) with CaCIPK1, -3, -6, -9, -14 and − 22, and exhibited weak (thinner lines) interactions with CaCIPK2, -4, -5, -7, -10, -13, -15, -18 and − 21 (Fig. 7). The CaCBL2 showed strong interactions with four CaCIPKs, including CaCIPK3, -6, -9 and − 22, and weak interactions with eight CaCIPKs (e.g. CaCIPK1, -2, -10, -13, -14, -15, -18 and − 21). CaCBL4 showed strong interactions with seven CIPKs, namely CaCIPK1, -2, -6, -9, -10, -14, -22. The CaCBL5 interacted with a total of 14 CaCIPKs among which it showed strong interactions with only CaCIPK9 and − 14 and weak interactions with CaCIPK1, -2, -3, -5, -6, -10, -13, -15, -16, -18, and − 22. The CaCBL8 was found to interact with nine CaCIPKs, including CaCIPK3, -5, -6, -7, -9, -14, -16, -21 and − 22. The CaCBL9 showed strong interaction with only two CaCIPKs namely CaCIPK9 and CaCIPK6, out of which CaCIPK9 shows homology with AtCIPK23 according to the phylogenetic tree. The CaCBL10 interacts with sixteen CaCIPKs (e.g. CaCIPK1, -2, -3, -4, -5, -6, -7, -9, -10, -13, 14, -15, 16, -18, -21, -22), out of those, it showed strong interactions with only two CaCIPKs (CaCIPK6 and CaCIPK9) (Table S6).
3.8. 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) (Fig. 8). The CaCIPK3, -4, -6, -7, -14, -15, -16, -18 and − 22 were found to have ubiquitously high expression in all the tissues. Whereas, CaCIPK2, -8, -13, -17, and − 21 showed low expression in almost all the tissues.
3.9. Expression profile in different stages of seed development
Optimum development of seeds leads to their production in sufficient quantity as well as quality, thereby determining the yield. To understand the role of CIPKs in chickpea seed development stages, 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) (Fig. 9). Few CaCIPK genes, including CaCIPK2, -11, -13 were ubiquitously expressed during all the seed stages in both the chickpea varieties, however, the level of expression varied (Table S8). CaCIPK2 expressed highly during S5-S7 in JGK3, and 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 in both varieties. Remarkably, CaCIPK6 and − 16 were upregulated during S1-S4 but downregulated during S5-S7 in both 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.
3.10 qRT-PCR expression analysis under ABA, drought and salinity stress
Abiotic stresses, such as drought and salinity cause the increase in cytosolic Ca2+ concentration, that develops a specific “Ca2+ signatures”. CIPKs are important components of Ca2+ sensing and signaling machinery in plants thus, they are crucial for plant’s adaptation to abiotic stresses. Therefore, to understand the role of CIPKs in abiotic stress signaling in chickpea, qRT-PCR analysis was performed for few selected genes including i.e., CaCIPK2, CaCIPK11, CaCIPK12, CaCIPK16, CaCIPK17 and CaCIPK21 under the treatment of abiotic stresses such as drought, salt stress and ABA. Expression data was obtained at different time points of treatment in root and shoot tissues, separately. Based on the expression fold change value ≥ 1.5 w.r.t. untreated control, all six CaCIPK genes showed differential expression under one or more stress conditions (Fig. 10; Table S9). In roots, all six genes expressed differentially under drought condition. CaCIPK2, 12, 17 and 21 were significantly up-regulated upon 1h, 3h and 6 hr of drought treatment, while CaCIPK11 was up-regulated after 1h and 3h of drought treatment. CaCIPK16 was induced after 3h of drought treatment, while it was down-regulated after 1h and 6h drought treatment. These six CaCIPK genes were also found to be differentially expressed under ABA treatment. CaCIPK2,11,12,16 and 21 were up-regulated at one or more time points. Interestingly, CaCIPK17 which was ubiquitously expressed under drought stress was found to be down-regulated under ABA treatment (Fig. 8).