Calcium-dependent Protein Kinases: Understanding Functional Diversification and Specificity of Plant Signalling Hubs Using the Most Conserved Members

Agricultural productivity and food security are declining globally, because of factors such as climate change, crop disease, natural calamities, population growth and pollution. Research on how to improve plant stress tolerance, disease resistance and crop productivity is paramount in preparing for future agro-environmental difficulties brought about by a changing world. Calcium-dependent protein kinases (CPKs) are plant proteins that directly bind calcium ions before phosphorylating substrates involved in osmosis, hormone response, stress and pathogen signalling pathways. CPKs are considered as ‘hubs’ in plant signalling; members of this large multigene family may function redundantly or complementarily to multiple stresses and stimuli. This research project aimed to answer three questions about the functional diversification and specificity of CPKs. Firstly, how did CPKs diversify and what is the most conserved CPK group in plants? Secondly, what is the role of the most conserved CPKs in plant stress and pathogen responses? Lastly, what influences CPK functional specificity? A comprehensive genome-wide phylogenetic analysis of CPKs from algae to higher plants showed that CPKs diversified in parallel with the transition of plants into terrestrial life, possibly providing support to plants in response to the stress of this transition; and that the most conserved members of this gene family in plants are those that belong to Group IIb. In Arabidopsis, CPKs that belong to this group are AtCPK3, 17 and 34. AtCPK3 and its orthologues (Group IIb.1) in rice and kiwifruit change in transcript accumulation in response to most abiotic stresses and pathogens such as Botrytis cinerea, Pseudomonas syringae, and various plant viruses, as inferred from meta-analysis of publicly available transcript data and as validated from biological experiments carried out in this project. Knocking out or overexpressing AtCPK3 in Arabidopsis and AcCPK16 in kiwifruit appeared to change the way the limited number of experimental plants respond to stress and pathogens. In Arabidopsis, overexpressors were slightly more tolerant to drought, bacterial, fungal and viral infections, whereas knockouts had little difference or were slightly more susceptible to WT. In kiwifruit, overexpressors were slightly more tolerant to drought and more susceptible to fungal infections, whereas knockouts had little difference or were slightly more susceptible to WT. AtCPK17 and 34 and their orthologues in rice and kiwifruit (Group IIb.2) were only expressed in floral tissue and mainly function in pollen development. Gene structure and predicted protein structure analysis of Group IIb CPKs in Arabidopsis and rice identified promoter regions and several protein motifs correlated to CPK function. Seed and pollen germination assays showed some degree of similarity in responses among AtCPK3 and AtCPK34 single overexpressors, suggesting that tissue localisation influences CPK gene function. Gene structure, several protein motifs and tissue localisation, all contribute to CPK functional specificity, which may explain why CPK functions are usually redundant and overlapping making them useful as plant signalling hubs. This project provides new insights and hypotheses with regards the evolution of CPKs and recommends further research with regards the use of group IIb.1 CPKs for novel molecular and diagnostic approaches in managing plant abiotic and biotic stress across a broad range of plant species