Identification of the AKCDPK gene family and AkCDPK15 functional analysis under drought and salt stress

Abstract

Konjac is one of the important economic crops for poverty alleviation in mountainous areas of Yunnan Province, China. However, there are always various biotic and abiotic stress during its growth, leading to production reduction and quality decline. Calcium-dependent protein kinases (CDPKs) are an important class of genes involved in calcium ion signal transmission within plant tissue cells, yet their presence and functions in konjac remain unexplored. This study aimed to identify the members of the AkCDPK gene family in the Amorphophallus konjac genome and understand their evolution and responses to various stresses. A total of 29 AkCDPK genes were identified and categorized into four subgroups that unevenly distributed across 12 chromosomes. Most AkCDPK have undergone purifying selection during evolution. Cis-acting element analysis revealed that several AkCDPK are involved in phytohormone induction, defence, stress response, and plant development. Expression analysis indicated tissue specificity, and responses to salt, drought, and Pectobacterium carotovorum subsp. carotovorum stress. AkCDPK15, encoding 582 amino acids, was cloned. AkCDPK15 was mainly localised on the cell membrane, and overexpression in tobacco revealed that it can positively regulate the tolerance of transgenic tobacco strains to salt and drought stress. These findings provide a theoretical foundation for future research on the function of the CDPK gene family in A. konjac, potentially aiding in the development of stress-resistant konjac varieties.

Fig 1. Phylogenetic relationships of CDPK proteins in Amorphophallus konjac (yellow pentagram), Arabidopsis thaliana (blue circle), and Oryza sativa (red box).

The evolutionary tree was constructed using MEGA10 software with the neighbour-joining method. The numbers next to the branch show 1000 bootstrap replicates, expressed as percentages. The phylogenetic groups of AkCDPK are marked with different colours and legends.

Fig 2. Phylogenetic relationships, gene structure, and conserved motifs of AkCDPK.

(A) Structure of the AkCDPK genes. Orange, black, and blue boxes represent the exons, introns, and untranslated regions, respectively. (B) Conserved motifs of AkCDPK proteins. The motifs are indicated by different coloured boxes and their numbers are listed on the right.

Fig 3. Physical map of AkCDPK chromosome locations.

The vertical axis represents the length of the chromosomes.

Fig 4. Synteny analysis of CDPK in Amorphophallus konjac, Arabidopsis thaliana, and Oryza sativa.

(A) Schematic representation of the chromosomal distribution and intrachromosomal relationships of A. konjac CDPK genes. (B) Collinear correlation between A. konjac and A. thaliana, and O. sativa. Grey lines in the background indicate the collinear blocks within A. konjac and other plant genomes, whereas the red lines highlight syntenic AkCDPK gene pairs.

Fig 5. Cis-acting regulatory elements in the AkCDPK promoter region.

Fig 6. Expression patterns of AkCDPK in different tissues (root, bulb, petiole and leaf).

The heat map shows the relative expression after log10 transformation.

Fig 7. Expression profiles of AkCDPK during Pectobacterium carotovorum subsp. carotovorum (Pcc) infection.

Fig 8. Expression profiles of AkCDPK under salt and mannitol stress.

Fig 9. Subcellular localisation of AkCDPK15.

Fig 10. Overexpression of AkCDPK15 improves tolerance of tobacco to drought and salt stress.

(A) AkCDPK15-overexpressing and wild-type strains grown for 14 days on standard 1/2 Murashige and Skoog (MS) medium supplemented with 200 mM NaCl. (B) The root length bar chart of AkCDPK15-overexpressing and wild-type strains grown for 14 days on standard 1/2 MS medium supplemented with 200 mM NaCl. (C) AkCDPK15-overexpressing and wild-type strains grown for 14 days on standard 1/2 MS medium supplemented with 100 mM mannitol. (D) The root length bar chart of AkCDPK15-overexpressing and wild-type strains grown for 14 days on standard 1/2 MS medium supplemented with 100 mM mannitol. (E) AkCDPK15-overexpressing and wild-type strains grown for 14 days on standard 1/2 MS medium without supplementation. (F) The root length bar chart of AkCDPK15 gene overexpression strains and wild-type strains grown for 14 days on standard 1/2 MS medium without supplementation. Different lowercase letters indicate significant differences, as calculated by Student’s t-test).

Fig 11. Phenotypes and physiological indices of AkCDPK15-overexpressing tobacco strains under normal and drought stress conditions.

(A) One-month-old WT strains grown under normal conditions. (B) AkCDPK15-overexpressing and WT strains after 14 days without water. (C) AkCDPK15-overexpressing and WT strains in (B) after re-watering for two days. (D-J) The content of malondialdehyde (MDA) (D), proline (E), soluble sugar (F), H₂O₂ (G) in WT and AkCDPK15-overexpressing strains under normal and drought stress. The activity of superoxide dismutase (SOD) (H), peroxidase (POD) (I), and catalase (CAT) (J) in WT and transgenic lines under normal and drought stress conditions. Data are presented as mean ± SD (n = 3, *P < 0.05; **P < 0.01, Student’s t-test).