Nitric oxide (NO) is an important cellular signalling molecule regulating various physiological processes, in both animals and plants. In animals, NO synthesis is mainly catalysed by NO synthase (NOS) enzymes. During biotic or abiotic stresses, NOSlike activities that are sensitive to mammalian NOS inhibitors have been detected in plants, although no sequences encoding the well described mammalian NOS are highlighted in land plants. Interestingly, we identified NOS-like sequences in 20 algae species. Among them, NOSs are found in Klebsormidium nitens the model alga to study the early transition step from aquatic algae to land plants. As mechanisms governing NO synthesis and signalling in green lineage remain unclear, the study of NOSs from K. nitens (KnNOS) through (i) characterization of their nucleotide and protein sequences, (ii) their expression levels, and (iii) the identification of partners proteins might be an important breakthrough knowledge. To achieve this goal, nucleotide sequences were verified by RACE-PCR and sequencing, and after cloning, tagged-proteins were produced. Then the identification of protein partners will be performed by pull-down experiments followed by mass spectrometry analysis. Currently, after re-annotating the nucleotide sequence of KnNOSs, a yeast His-tagged protein production strategy has been adopted. While waiting for the final results of the KnNOSs interactome, an in sillico study was conducted using the BioGrid database and human NOS interaction data. This finding opens the way for a deeper characterization of KnNOSs and its protein partners and will facilitate further investigation of NO signalling in green lineage.