Cold exposure of plants triggers an intracellular increase in the cytoplasmic calcium concentration. Calcium-dependent protein kinases (CDPK) have been discussed as calcium sensors that perceive and translate these changes into downstream signalling to rapidly induce appropriate cold stress responses. The objective of project A4 is to assess a role of CDPKs in cold priming of Arabidopsis thaliana, whereby a priming exposure to low, non-freezing temperatures prepares the plant for improved tolerance to frost (subzero temperature) which the plant experiences after a longer period of time over days and weeks after the priming cold experience. In phase I we have established a robust priming protocol, and molecular, metabolic and morphological changes during this protocol have been investigated. In line with our hypothesis cold primed plants display a higher survival rate under freezing conditions. Our analysis identified so far one distinct CDPK isoform as negative regulator of cold priming: The corresponding cpk mutant reveals increased frost tolerance upon priming / and triggering, indicating its contribution to priming and memory, the generated overexpressing line shows reduced survival.
Our current analysis investigates how these cpk-dependent phenotypes coincide with altered levels of key marker metabolites including sugars, amino acids, and lipids that are described in the context of cold acclimation.
Research in phase II will investigate firstly the biochemical regulation of this CPK isoform. We will address whether calcium sensitivity respectively calcium-regulated kinase activity of the enzyme, which functions as a negative regulator, influences onset, strength and duration of priming and memory - or of forgetting. This will be complemented by MS-based measurement of the in vivo phosphorylation status of the kinase during the priming protocol. Secondly, we will conduct an expression analysis (RNAseq) and select for a cluster of co-expressed genes that are transcriptionally enhanced in the cpk mutant during the priming protocol. Based on promoter sequences of selected genes, transcription factors regulating these genes (and hence representing potential CPK targets) will be identified using the yeast one-hybrid system (collaborative work). In an eco-physiological approach the role of the CPK (orthologue) will be assessed in a collection of Arabidopsis accessions of different geo-botanical origins, where CPK expression will be correlated to priming-dependent freezing tolerance (collaborative work).