Molecular Characterization Reveals the Involvement of Calcium Dependent Protein Kinases in Abiotic Stress Signaling and Development in Chickpea (Cicer arietinum)

Abstract

Calcium-dependent protein kinases (CDPKs) are a major group of calcium (Ca²⁺⁾ sensors in plants. CDPKs play a dual function of "Ca²⁺ sensor and responder." These sensors decode the "Ca²⁺ signatures" generated in response to adverse growth conditions such as drought, salinity, and cold and developmental processes. However, knowledge of the CDPK family in the legume crop chickpea is missing. Here, we have identified a total of 22 CDPK genes in the chickpea genome. The phylogenetic analysis of the chickpea CDPK family with other plants revealed their evolutionary conservation. Protein homology modeling described the three-dimensional structure of chickpea CDPKs. Defined arrangements of α-helix, β-strands, and transmembrane-helix represent important structures like kinase domain, inhibitory junction domain, N and C-lobes of EF-hand motifs. Subcellular localization analysis revealed that CaCDPK proteins are localized mainly at the cytoplasm and in the nucleus. Most of the CaCDPK promoters had abiotic stress and development-related cis-regulatory elements, suggesting the functional role of CaCDPKs in abiotic stress and development-related signaling. RNA sequencing (RNA-seq) expression analysis indicated the role of the CaCDPK family in various developmental stages, including vegetative, reproductive development, senescence stages, and during seed stages of early embryogenesis, late embryogenesis, mid and late seed maturity. The real-time quantitative PCR (qRT-PCR) analysis revealed that several CaCDPK genes are specifically as well as commonly induced by drought, salt, and Abscisic acid (ABA). Overall, these findings indicate that the CDPK family is probably involved in abiotic stress responses and development in chickpeas. This study provides crucial information on the CDPK family that will be utilized in generating abiotic stress-tolerant and high-yielding chickpea varieties.

Keywords: CDPK; abiotic stress; chickpea; development; gene expression; signaling.

Figure 1

Structural features of the chickpea calcium-dependent protein kinases (CDPK) family. (A) Exon-intron organization is shown for CaCDPK genes and gene names are mentioned at the left. The scale at the bottom represents gene length in kb. (B) Protein domain structure of the chickpea CDPK family is showing conserved protein kinase and EF-hand along with some important sites present. ATP binding site and Serine/threonine-protein kinase active site are located at the protein kinase domain.

Figure 2

Phylogenetic relationship among CDPKs from different plants. An un-rooted neighbor-joining phylogenetic tree was constructed from the CDPK protein sequences of Arabidopsis thaliana, rice, soybean, and chickpea. All the CDPKs could be divided into four groups; I- IV. Numbers above the nodes represent bootstrap values calculated from 1,000 replicates.

Figure 3

Chromosomal locations of chickpea CDPK genes. Green color bars represent the chromosomes, the location of genes has been marked alongside. Chromosome numbers are given at the bottom of each chromosome. Except chromosome 1, the CDPK genes are distributed variably on seven chromosomes.

Figure 4

Three-dimensional structure of chickpea CDPK proteins. A three-dimensional (3-D) structure was generated for all 22 members of the chickpea CDPK family. Each CDPK protein is made up of a variable number of α-helix, β-strands, transmembrane helix, and disordered region. Structure comprising blue and green α-helices and β-strands represents kinase domain, yellow helical ribbons represent inhibitory junction domain (JD), orange helices indicate N-lobe, and red helices indicate C-lobe with each containing two elongation factors (EF) hand motifs.

Figure 5

Subcellular localization of chickpea CDPK proteins. (A) Localization was predicted using an online tool, CELLO. The figure shows chickpea CDPK proteins localized majorly in the cytoplasm and nucleus based on the highest reliability score with black color. Proteins that were predicted to be localized both in the cytoplasm and nucleus are shown by blue color at both locations. (B) N. benthamiana cells expressing the YFP- fusion protein driven by the 2XCaMV 35S promoter. Confocal images of fluorescence expressing CaCDPK proteins are showing their variable distribution in different compartments. CaCDPK5 is localized to the cytoplasm and nucleus, whereas CaCDPK16 and CaCDPK21 are localized at the cytoplasm. Cells transformed with vector only (CaMV35S-YFP) are shown in the lowermost row. Scale bar = 20 μm.

Figure 6

Promoter analysis of chickpea CDPK genes. Different cis-regulatory elements in the 2 kb upstream promoter region of CaCDPK genes are illustrated by different colors in the bar chart. The X-axis represents the name of genes and Y-axis represents the number of different cis-elements in each promoter. The names of cis-regulatory elements are mentioned on the right.

Figure 7

Expression profiles of CDPK genes in different developmental stages of chickpea. The heatmap represents the expression pattern of CaCDPK genes in different tissues of developmental stages, such as germination, seedling, vegetative, reproduction, and senescence. The genes are named on the left and different tissues/developmental stages are labeled at the top. Scale bar represents the normalized log_2 FPKM values.

Figure 8

Expression profiles of CDPK genes during different stages of seed development in chickpea. Expression profiles are shown for seven seed developmental stages (S1–S7) in two chickpea cultivars: large-seeded JGK3 and small-seeded Himchana1. The genes are named on the left and stages/cultivars are labeled at the top. The scale bar represents the log_2 Fold Change FPKM values.

Figure 9

Expression profiles of CDPK genes under drought, salt, and ABA treatments in chickpea. qRT-PCR analysis was performed to generate the expression profile of CaCDPK genes under drought, salt, and ABA treatment in root and shoot of desi chickpea (ICC4958). Different treatments and time points are indicated on X-axis and the relative expression level of the gene is indicated on Y-axis. Each bar represents the mean value of three replicates. Standard error among the samples is indicated by error bars. *p < 0.05, **p < 0.01 and ***p < 0.005 for treated samples w.r.t. untreated control.