Ive De Smet leads the Functional Phosphoproteomics Group. To fully understand plant growth and development, we need to identify novel components and require insight in the underlying network. In this context, there is an urgent need to gain insight in protein changes on different levels, including protein-protein interactions and post-translational protein modifications. With respect to the latter, temporary and reversible phosphorylation of proteins is essential in regulating intracellular biological processes. Phosphorylation affects protein folding (conformation), protein function and the regulation of enzymatic activities, defines substrate specificity, and influences protein localization, complex formation and degradation. The mechanistic importance of phosphorylation is obvious from its major influence on various cell functions, such as signal transduction, cell division, cell differentiation, and metabolic maintenance.
As a biological model, the Functional Phosphoprotemics Group focuses on thermomorphogenesis, a process whereby plants respond to mild warm temperature conditions by increased elongation growth of organs to enhance cooling capacity. Although our understanding of temperature perception and response in plants has increased in recent years, we still know relatively little about the cellular signalling cascades that control architectural adaptations to high ambient temperatures. Arabidopsis thaliana has been proven to be an efficient model plant to study plant growth and development, but time has come to investigate signaling cascades in crop species, such as wheat and soybean, species that are currently also under investigation.
Almost every organism is exposed to variation in temperature, on a daily and on a seasonal basis. This is especially true for plants that, as sessile organisms, need to continuously alter their growth, development, and physiology in response to temperature variation. To sense and respond to temperature changes, several molecular sensors and downstream signalling and response networks have evolved. Although our understanding of temperature perception and response in plants has increased in recent years, we still know relatively little about the cellular signalling cascades that control architectural adaptations to high ambient temperatures (referred to as thermomorphogenesis), which allow improved cooling by evaporation to withstand warm temperatures.
Phosphatases and Kinases
While the knowledge on post-translational regulation through transient phosphorylation in plants is growing because of its crucial importance in plant molecular networks, it remains an underexplored and challenging area.
In Arabidopsis and major crop species, phosphorylation is controlled by a large number of protein kinases and phosphatase complexes. However, for the majority of cytoplasmic kinases, membrane-associated receptor kinases and phosphatases unravelling physiological and developmental roles and identifying substrates remain a challenge.
We apply a gel-free phosphoproteomics pipeline to different biological systems: wheat and soybean organs, Arabidopsis cell suspension cultures, and Arabidopsis seedlings. We combine these systems with loss- and gain-of-function approaches (such as tightly controlled systems using a constitutively active form under a native, inducible promoter), engineered kinases, and specific stimuli to perform an untargeted mass spectrometric analysis of the phosphoproteome. To confirm the importance of key differentially phosphorylated proteins (ideally hubs controlling major switches in physiological and developmental processes) in a biological process, candidates are functionally characterized using kinase assays and phospho-specific antibodies to demonstrate functionality and in vivo phosphorylation, respectively, complemented with loss- and gain-of-function approaches (including site-directed phospho-site mutagenesis) and detailed analyses of expression patterns, both in Arabidopsis and monocot crops when relevant.