Nitric oxide (NO) is an important cellular signaling molecule across kingdoms. During biotic or abiotic stresses, NO burst is detected in both plants and mammals although no sequence encoding the well described mammalian NO synthase (NOS) is highlighted in plants. Compared to terrestrial plants, some algae present transcripts encoding the NOS-like enzyme. Among them, Klebsormidium nitens the model alga to study the early transition step from aquatic algae to land plants is found. As mechanisms governing NO synthesis and signaling in green lineage remain unclear, the study of NOS from K. nitens (KnNOS) through (i) the identification of regulator proteins, (ii) the identification of target proteins, and (iii) their expression levels might be an important breakthrough knowledge. To achieve this goal, the identification of protein partners will be performed by pull down experiments followed by mass spectrometry analysis. Despite the KnNOS is a challenging protein to produce and purify, we are currently optimizing a promising protocol to obtain recombinant GST-tagged KnNOS protein in Escherichia coli. Since expression level analysis require suitable reference genes for reliable normalization, we selected 13 candidate reference genes based on literature, and performed RT-qPCR to study expression stability during 4 abiotic stresses commonly used. Final ranking list of most stable reference genes was obtain based on results from algorithms including, the ΔCt method, geNorm, NormFinder and BestKeeper. This finding opens the way for a deeper characterization of KnNOS and its protein partners and will facilitate further investigation of gene expression in K. nitens.