Begum, K.; Das, A.; Ahmed, R.; Akthar, S.; Banu, S.

Calcium is the most common secondary messenger of the plant signal transduction mechanism. The Calcium-dependent protein kinases (CDPKs), a plant multi-gene family protein, act as sensors of calcium ion concentration inside plant cells. CDPKs convert variations in calcium concentration into a signal and phosphorylate various proteins that translate calcium signals into physiological reactions. Aquilaria agallocha is an aromatically important crop due to the production of valuable fragrant resinous agarwood in their heartwood. The trees produce agarwood when wounded and exposed to biotic and abiotic stresses. CDPKs are essential in plant development and growth, biotic and abiotic stress responses, and phytohormone-mediated signalling pathways. A comprehensive investigation of the stress-combating gene family (CDPK) in the A. agallocha genome is currently lacking. In this work, we used a bioinformatics approach to examine the entire genome of A. agallocha and identified 24 CDPK genes. According to the molecular phylogenetic relationships, the putative AaCDPKs are grouped into four groups. Synteny analysis identified a few conserved segments (orthologous genes) between A. agallocha and Arabidopsis thaliana (eight pairs), A. sinensis (twenty pairs), Glycine max (sixteen pairs), Solanum tuberosum (five pairs), and Vitis vinifera (ten pairs). Duplication analysis of AaCDPK genes indicated that dispersed duplications significantly contributed to the expansion of the CDPK gene family of A. agallocha. The observed AaCDPK-RBOH interaction within the protein interaction network suggests that this interaction may be crucial in integrating Ca2+ and ROS signaling pathways. RNA-seq data analysis shows differential expression of seven putative AaCDPK genes in agarwood tissue. qRT-PCR analysis revealed that six out of ten selected AaCDPK genes exhibited altered expression levels in response to MeJA, H2O2, and CaCl2 treatments. Integrated promoter analysis, PPI data, and in silico gene expression validated with qRT-PCR-analysis collectively suggest the role of CDPK-RBOH as a signalling molecule in initiating phytohormone-mediated agarwood resin formation. This study provides a strong foundation for future investigations into the study of functional roles and regulatory mechanisms of AaCDPK genes in sesquiterpene biosynthesis and agarwood formation.