Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens, a model alga to study plant adaptation to land

National audienceThe small gaseous molecule nitric oxide (NO) is well established as a major ubiquitous component of cell signalling. A key signalling mechanism mediating NO effects is S-nitrosation, a post-translational modification by which NO can impact the target protein activities, subcellular localizations, and capacities to form protein complexes. The identification of proteins targeted by NO is of major interest in order to elucidate NO functions. Interestingly, land plants lack NO synthase (NOS), which is the main enzyme for NO synthesis in metazoans, while a few algal species possess it, thus raising many interrogations. Therefore, we focused on the identification of S-nitrosated proteins during salt stress in Klebsormidium nitens, a freshwater algal species possessing a NOS and established as a model to study plants adaptation to land. We applied the Biotin Switch method followed by mass spectrometry analysis. This method allows the purification of S-nitrosated proteins. We found 43 proteins with significantly higher S-nitrosation levels in salt response condition. Orthology analysis were performed against the model plant Arabidopsis thaliana, therefore we established a classification of these proteins according to their localization, molecular function and biological process they are involved in. This is done with the PANTHER classification system, based on Gene Ontology terms (GO terms). Then, from this list, we selected an interesting protein called INOSITOL POLYPHOSPHATE MULTIKINASE 2a/b (IPK2a/b), potentially involved in cell signaling and stress response. We produced it in Escherichia coli and are currently measuring its activity. This protein will then be Snitrosated in vitro and we will determine if its activity is impaired. Through the identification and functional analysis of Snitrosated proteins in K. nitens, this project will provide a better understanding of the functions of NO in unicellular green algae with NO