Role of nitric oxide synthases from Klebsormidium nitens: first structural characterization and partners identification
Résumé
Objectives: Nitric oxide (NO) is an important cellular signaling molecule regulating various physiological
processes, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase
(NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have been
measured, although no sequences encoding mammalian NOSs have been found in land plants.
Interestingly, we identified NOS-like sequences in 20 algae species. These latter include the
filamentous charophyte green algae Klebsormidium nitens, a biological model to study the early
transition step from aquatic algae to land plants. In order to understand the mechanisms governing
NO synthesis and signaling in green lineage we initiated the functional characterization of K. nitens
NOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response to
abiotic stresses.
Methods: KnNOSs nucleotide sequences were verified by RACE-PCR and sequencing, and their mRNA
level were monitored by RT-qPCR and protein abundance by western blot. Protein partners were
studied, firstly in sillico using the BioGrid database and human NOS interaction data, and secondly in
vivo by immunoprecipitation experiments followed by mass spectrometry analysis.
Results: Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possesses
classical mammalian NOS architecture consisting of oxygenase and reductase domains with some
specificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2
displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seem
to be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditions
tested, whereas KnNOS2 appeared to be transcriptionally regulated during stress. In parallel studies,
we also built the in silico protein–protein interaction network of human NOSs. Interestingly, genes
encoding orthologs of several of these candidates were found in K. nitens genome. Some of these
conserved partners are known to be involved in mammalian NOSs regulation and represent interesting
candidates for further investigation.
Conclusions: Overall these findings open the way for a deeper characterization of KnNOSs and its
protein partners and will facilitate further investigation of NO signaling in green lineage.
Relevant references:
Chatelain, P., Astier, J., Wendehenne, D., Rosnoblet, C., and Jeandroz, S. (2021). Identification of Partner Proteins
of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic
Photosynthetic Organisms. Front. Plant Sci. 12, 3068. doi: 10.3389/fpls.2021.797451.
Jeandroz, S., Wipf, D., Stuehr, D. J., Lamattina, L., Melkonian, M., Tian, Z., et al. (2016). Occurrence, structure,
and evolution of nitric oxide synthase–like proteins in the plant kingdom. Sci. Signal. 9, re2–re2. doi:
10.1126/scisignal.aad4403.