Functional dissection of novel protein kinases and their roles in diurnally regulated signal transduction in Arabidopsis thaliana

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Plants utilize light energy to harvest carbon dioxide from the air to build complex molecules. The ability to detect (light signaling) and/or anticipate (circadian clock) the availability of light represents a major determinant of plant growth and survival. Together, these abilities form a diurnal regulatory circuit which allows plants to dynamically adapt to their daily environment. At the molecular level, diurnal regulation is achieved through a combination of transcriptional, translational and post-translational mechanisms. In particular, post-translational protein modifications (PTMs), which are central to the proper functionality of eukaryotic cells. PTMs represent a fast-acting, fine-tuning mechanism for proteins that can have diverse outcomes such as a change in enzyme activity or cellular localization. Of the PTMs, protein phosphorylation is the most abundant, regulating over 70% of all eukaryotic proteins. However, with our current understanding of diurnal plant cell regulation largely derived from changes in transcript levels, the extent of regulation by protein level and PTM changes remains poorly understood. The long term goal of my research is to utilize quantitative proteomics to uncover new diurnal signaling proteins in the model plant Arabidopsis and to understand how these proteins function to regulate plant cell processes. Currently, our understanding of how protein phosphorylation diurnally regulates cellular processes is limited to a handful of studies. Furthermore, connections between diurnal phosphorylation events and the protein kinases responsible for catalyzing these events is limited. Using phosphoproteomics, we have recently shown major quantitative differences exist in the end-of-day and end-of-night phosphoproteome of Arabidopsis thaliana across multiple tissue types. Here, we identified a number of protein kinases undergoing diurnal changes in their phosphorylation status; in particular, an unique set of plant specific Phox/BemP1 domain containing protein kinases (PXKs). Given the diverse outcomes phosphorylation events can have in controlling protein function, we hypothesize that the diurnal phosphorylation changes we observed through our multi-tissue phosphoproteomic screen are indicative of each PXKs broader involvement in the regulation of diurnal plant cell processes. To evaluate this, we will (1) undertake biochemical and biological characterization of the PXK sub-family, (2) identify and quantify diurnal PXK protein interaction networks and (3) undertake functional characterization of PXK protein complexes. Together, these 3 objectives will uncover the role each PXK plays in diurnal plant cell regulation, while also helping to further bridge the gap that exists between plant phosphoproteomics and the specific protein kinases that catalyze these reactions.