Role of nitric oxide synthases from Klebsormidium nitens: first structural characterization and partners identification

Abstract

International audienceObjectives: Nitric oxide (NO) is an important cellular signaling molecule regulating various physiologicalprocesses, 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 beenmeasured, 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 thefilamentous charophyte green algae Klebsormidium nitens, a biological model to study the earlytransition step from aquatic algae to land plants. In order to understand the mechanisms governingNO synthesis and signaling in green lineage we initiated the functional characterization of K. nitensNOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response toabiotic stresses.Methods: KnNOSs nucleotide sequences were verified by RACE-PCR and sequencing, and their mRNAlevel were monitored by RT-qPCR and protein abundance by western blot. Protein partners werestudied, firstly in sillico using the BioGrid database and human NOS interaction data, and secondly invivo by immunoprecipitation experiments followed by mass spectrometry analysis.Results: Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possessesclassical mammalian NOS architecture consisting of oxygenase and reductase domains with somespecificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seemto be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditionstested, 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, genesencoding orthologs of several of these candidates were found in K. nitens genome. Some of theseconserved partners are known to be involved in mammalian NOSs regulation and represent interestingcandidates for further investigation.Conclusions: Overall these findings open the way for a deeper characterization of KnNOSs and itsprotein 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 Proteinsof the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in EukaryoticPhotosynthetic 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