Identification of Partner Proteins of the Algae Klebsormidium nitens NO Synthases: Toward a Better Understanding of NO Signaling in Eukaryotic Photosynthetic Organisms

Abstract

In animals, NO is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS) enzyme. NO production and effects have also been reported in plants but the identification of its sources, especially the enzymatic ones, remains one of the critical issues in the field. NOS-like activities have been reported, although there are no homologs of mammalian NOS in the land plant genomes sequenced so far. However, several NOS homologs have been found in algal genomes and transcriptomes. A first study has characterized a functional NOS in the chlorophyte Ostreococcus tauri and the presence of NOS homologs was later confirmed in a dozen algae. These results raise the questions of the significance of the presence of NOS and their molecular diversity in algae. We hypothesize that comparisons among protein structures of the two KnNOS, together with the identification of their interacting partner proteins, might allow a better understanding of the molecular diversification and functioning of NOS in different physiological contexts and, more generally, new insights into NO signaling in photosynthetic organisms. We recently identified two NOS homologs sequences in the genome of the streptophyte Klebsormidium nitens, a model alga in the study of plant adaptation to terrestrial life. The first sequence, named KnNOS1, contains canonical NOS signatures while the second, named KnNOS2, presents a large C-ter extension including a globin domain. In order to identify putative candidates for KnNOSs partner proteins, we draw the protein–protein interaction networks of the three human NOS using the BioGRID database and hypothesized on the biological role of K. nitens orthologs. Some of these conserved partners are known to be involved in mammalian NOSs regulation and functioning. In parallel, our methodological strategy for the identification of partner proteins of KnNOS1 and KnNOS2 by in vitro pull-down assay is presented.

Reliable reference genes and abiotic stress marker genes in Klebsormidium nitens

“…Due to its importance as a model organism, studies using K. nitens have been increasingly published and have been reported investigations focusing, for instance, on the evolution of auxin 14 , jasmonate 28 , nitric oxide signaling 29 , the origin of mycorrhiza 15 , or the response to high light stress 30 . Transcriptomic approaches using RT-qPCR are commonly used, because of their simplicity and low cost for laboratories 21 .…”

“…ICE-L 26 , Ulva linza 22 or Chorella pyrenoidosa 27 . However, despite the growing interest for K. nitens 14,15,[28][29][30] in the scientific community, no studies have been dedicated to the search of reference genes in this algal species. Nevertheless, the identification of best reference genes for normalization of RT-qPCR data is definitively required for further analyses.For this purpose, we determined the best couples of reference genes in K. nitens in response to three abiotic stresses: a high salt content, PEG-induced dehydration and heat shock.…”

Arginine-Dependent Nitric Oxide Generation and S-Nitrosation in the Non-Photosynthetic Unicellular Alga Polytomella parva

“…In photosynthetic plants, NR, mitochondrial electron transport chain, NOS-like enzymes, and putative polyamine oxidases have been suggested as sources of NO [6,7,43,44]. In contrast to embryophytes, several NOS homologs were found in algal genomes and transcriptomes [11,12,45]. In searching for NO sources, P. parva treatment with the mammalian NOS inhibitors L-NAME and AG was found to reduce the NO levels in cells incubated with arginine (Figure 3).…”

“…These data confirmed the role of arginine in NO production in P. parva. bryophytes, several NOS homologs were found in algal genomes and transcriptomes [11,12,45]. In searching for NO sources, P. parva treatment with the mammalian NOS inhibitors L-NAME and AG was found to reduce the NO levels in cells incubated with arginine (Figure 3).…”

Arginine as the sole nitrogen source for Ostreococcus tauri growth: Insights on nitric oxide synthase enzyme

“…Besides N source, the latest data have described NO as a signal molecule in algae (Foresi et al, 2010;Foresi et al, 2017;Astier et al, 2021;Chatelain et al, 2021). NO acts as a signal in the transcriptional and posttranslational regulation of NR and inorganic N transport in Chlamydomonas (Sanz-Luque et al, 2015).…”