Insight into the CBL and CIPK gene families in pecan (Carya illinoinensis): identification, evolution and expression patterns in drought response

Abstract

Background: Calcium (Ca²⁺) serves as a ubiquitous second messenger and plays a pivotal role in signal transduction. Calcineurin B-like proteins (CBLs) are plant-specific Ca²⁺ sensors that interact with CBL-interacting protein kinases (CIPKs) to transmit Ca²⁺ signals. CBL-CIPK complexes have been reported to play pivotal roles in plant development and response to drought stress; however, limited information is available about the CBL and CIPK genes in pecan, an important nut crop.

Results: In the present study, a total of 9 CBL and 30 CIPK genes were identified from the pecan genome and divided into four and five clades based on phylogeny, respectively. Gene structure and distribution of conserved sequence motif analysis suggested that family members in the same clade commonly exhibited similar exon-intron structures and motif compositions. The segmental duplication events contributed largely to the expansion of pecan CBL and CIPK gene families, and Ka/Ks values revealed that all of them experienced strong negative selection. Phylogenetic analysis of CIPK proteins from 14 plant species revealed that CIPKs in the intron-poor clade originated in seed plants. Tissue-specific expression profiles of CiCBLs and CiCIPKs were analysed, presenting functional diversity. Expression profiles derived from RNA-Seq revealed distinct expression patterns of CiCBLs and CiCIPKs under drought treatment in pecan. Moreover, coexpression network analysis helped to elucidate the relationships between these genes and identify potential candidates for the regulation of drought response, which were verified by qRT-PCR analysis.

Conclusions: The characterization and analysis of CBL and CIPK genes in pecan genome could provide a basis for further functional analysis of CiCBLs and CiCIPKs in the drought stress response of pecan.

Keywords: CBL-CIPK; Carya illinoinensis; Drought; Evolution; Gene expression.

Fig. 1

Phylogenetic relationship, gene structure and motif organization analyses of CBLs (A) and CIPKs (B) from pecan and Arabidopsis. The phylogenetic trees on the left included 19 CBL and 56 CIPK proteins from pecan and Arabidopsis and are divided into different clades. The conserved motifs are represented by different coloured boxes. Exon/intron structures of CBL and CIPK genes from pecan are also shown. Green boxes: untranslated regions; yellow boxes: exon regions; black lines: introns

Fig. 2

Domain analysis of pecan CBL proteins. Multiple sequence alignment was performed by ClustalW and illustrated by GeneDoc. The four EF-hand motifs were marked by overbars

Fig. 3

Circos figure for chromosomal locations and interchromosomal relationships of CBL and CIPK genes from pecan. The CIPK genes are marked in blue, and the CBL genes are marked in orange. The blue lines in the background represent the gene pairs in the CIPK family, and the orange lines indicate the gene pairs in the CBL family

Fig. 4

Phylogenetic relationships of the CBL (A) and CIPK (B) proteins of pecan with those of 13 plant species. The full-length CBL and CIPK protein sequences from C. illinoinensis, C. cathayensis, J. regia, A. thaliana, B. braunii, M. polymorpha, P. patens, S. moellendorffii, A. trichopoda, O. sativa, A. comosus, B. platyphylla, P. trichocarpa, and S. purpurea were used to build the phylogenetic tree with FastTree. The algae CBL and CIPK proteins were applied as outgroups. The clades are highlighted in different colours

Fig. 5

Hierarchical clustering of expression levels of pecan CBL and CIPK genes in different tissues. A Expression patterns of CBL genes in various pecan tissues. B Expression patterns of CIPK genes in various pecan tissues. Heatmaps of CiCIPK and CiCBL genes were built based on log ₁₀ (FPKM+ 1) values using R language

Fig. 6

Hierarchical clustering of expression levels of pecan CBL and CIPK genes in response to drought. A Expression patterns of CBL genes under drought. B Expression patterns of CIPK genes under drought. Heatmaps of CiCIPK and CiCBL genes were built based on log ₁₀ (FPKM+ 1) values using R language

Fig. 7

Expression analysis of CiCBL and CiCIPK genes in pecan exposed to drought stress. Expression patterns of 5 CiCBL (A) and 10 CiCIPK (B) genes were analysed using qRT–PCR. The actin gene (CiPaw.03G124400) was selected as the reference gene. Lowercase letters represent significant differences (P < 0.05) according to Duncan’s multiple range test. Error bars indicate the means ± SE obtained from three biological replicates

Fig. 8

Subcellular localization of three CiCIPKs based on the transient expression of fused GFPs. Control, GFP alone. Bar = 10 μm