Genome-Wide Identification and Characterization of CDPK Gene Family in Cultivated Peanut (Arachis hypogaea L.) Reveal Their Potential Roles in Response to Ca Deficiency

Abstract

This study identified 45 calcium-dependent protein kinase (CDPK) genes in cultivated peanut (Arachis hypogaea L.), which are integral in plant growth, development, and stress responses. These genes, classified into four subgroups based on phylogenetic relationships, are unevenly distributed across all twenty peanut chromosomes. The analysis of the genetic structure of AhCDPKs revealed significant similarity within subgroups, with their expansion primarily driven by whole-genome duplications. The upstream promoter sequences of AhCDPK genes contained 46 cis-acting regulatory elements, associated with various plant responses. Additionally, 13 microRNAs were identified that target 21 AhCDPK genes, suggesting potential post-transcriptional regulation. AhCDPK proteins interacted with respiratory burst oxidase homologs, suggesting their involvement in redox signaling. Gene ontology and KEGG enrichment analyses affirmed AhCDPK genes' roles in calcium ion binding, protein kinase activity, and environmental adaptation. RNA-seq data revealed diverse expression patterns under different stress conditions. Importantly, 26 AhCDPK genes were significantly induced when exposed to Ca deficiency during the pod stage. During the seedling stage, four AhCDPKs (AhCDPK2/-25/-28/-45) in roots peaked after three hours, suggesting early signaling roles in pod Ca nutrition. These findings provide insights into the roles of CDPK genes in plant development and stress responses, offering potential candidates for predicting calcium levels in peanut seeds.

Keywords: CDPK; calcium deficiency; calcium-dependent protein kinases; cultivated peanut; gene expression; stress response.

Figure 1

Chromosome density and chromosomal distribution of the AhCDPK genes. Chromosome numbers are provided at the left of each chromosome.

Figure 2

Synteny relationship of AhCDPKs in cultivated peanut. The blue lines indicate tandem duplicated gene pairs, and the red lines indicate segmentally duplicated and WGD gene pairs. The gray lines indicate segmentally duplicated gene pairs within the peanut genome. The scale bar marked on the chromosome indicates chromosome lengths (Mb).

Figure 3

Synteny analysis of CDPK genes between cultivated peanut and its two diploid ancestors (A) and among peanut, rice, and Arabidopsis (B). Gray lines in the background indicate the collinear blocks within peanut and other plant genomes, while the red lines highlight the syntenic CDPK gene pairs.

Figure 4

Phylogenetic analysis of CDPK proteins. The proteins of peanut, Arabidopsis, rice, Arachis duranensis, and Arachis ipaensis are represented in red star, green triangle, blue square, dark green circle, and dark red circle, respectively.

Figure 5

Conserved motifs analyses of AhCDPK proteins. (A) Conserved motif by MEME. The colored boxes on the right denote motifs 1–10. (B) Pfam motif. (C) SMART motif. (D) PROSITE profile.

Figure 6

Gene structure analyses of AhCDPK genes. The green boxes, black lines, and yellow boxes represent UTR, introns, and CDS, respectively.

Figure 7

Prediction of AhCDPK proteins’ tertiary structure by AlphaFold2 method. Models were visualized using rainbow colors from N to C terminus.

Figure 8

Cis-regulatory elements analysis of AhCDPK genes upstream regions. (A) The numbers and the depth of red represent the frequency of the elements that occur in the promoter region. (B) The statistics of categories on every AhCDPK. Different categories are present with different colors. (C) The number of each identified element.

Figure 9

Network map of projected miRNA targeting AhCDPK genes. The green boxes correspond to AhCDPK genes, and the pink boxes indicate predicted miRNAs.

Figure 10

Predictive interaction network of CDPK proteins in peanut. Network nodes represent proteins, and edges represent protein–protein associations.

Figure 11

GO (A) and KEGG (B) enrichment analysis of AhCDPKs.

Figure 12

Expression patterns of AhCDPK genes in different developmental tissues. (A) RNA-seq analysis. The heatmap was constructed using the mean of 1, 2, or 3 biological replicates, details in Table S1. The labels A, B, and C in the heatmap were shortened to indicate Cluster A, Cluster B, and Cluster C, respectively. (B) RT-qPCR analysis. Data are means ± SD (n = 6). Different letters indicate significant differences (Duncan’s test, p < 0.05) in each tissue including the subgraph.

Figure 13

Expression patterns of CDPK genes in peanut in response to abiotic stresses. (A) RNA-seq analysis. The heatmap was constructed using the mean of three biological replicates. RT-qPCR analysis when exposed to drought (B), salt (C), and cold (D). Data are means ± SD (n = 6). Different letters indicate significant differences (Duncan’s test, p < 0.05).

Figure 14

Expression patterns of CDPK genes in peanut pods in response to Ca deficiency. (A) RNA-seq analysis. The heatmap was constructed using the mean of three biological replicates. RT-qPCR analysis in (B) leaf and (C) root. Data are means ± SD (n = 6). Different letters indicate significant differences (Duncan’s test, p < 0.05).