CONICET | Buscador de Institutos y Recursos Humanos

Nitric oxide (NO) functions as a signalling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-Arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, and there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported four years ago. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage. In this chapter we describe with detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of it activity or the used, fluorescent probes, and Griess assay for in vivo determination. We further discuss the discussing their advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organism that can generate misunderstandings in for the interpretation of results.