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

Pauline Chatelain,Claire Rosnoblet,Sylvain Jeandroz,David Wendehenne,Jeremy Astier

doi:10.3389/fpls.2021.797451

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.

Highlights

Nitric Oxide and Its BiosynthesisNO is a ubiquitous signaling gaseous molecule which regulates a wide array of physiological, biochemical, and molecular events in animals, plants, and microbes
NO is mainly synthetized from L-arginine by three isoforms of nitric oxide synthases (NOSs, EC 1.14.13.39): neuronal NOS or NOS1, inducible NOS or NOS2, and endothelial NOS or NOS3 (Stuehr et al, 2004)
KnNOS2, The “NOS-Globin” Model we identified in the K. nitens genome a second putative NOS sequence showing a large C-ter extension of approximately 380 residues (GAQ90485.1)

Summary

INTRODUCTION

NO is a ubiquitous signaling gaseous molecule which regulates a wide array of physiological, biochemical, and molecular events in animals, plants, and microbes. The corresponding molecular actors, those related to NO and cGMP synthesis, remain to be identified to confirm the existence of a GA/NO/cGMP signaling cascade in rice Homologs of this rice PKG are generally present in single copy and well distributed among the green lineage, in algae and land plants. Thereby, our preliminary in silico approach highlights the promising presence of two NOSs and putative numerous NOS interacting partners in the genome of K. nitens We hypothesize that these enzymes would participate in a complex signaling networks, as the ones described in animals, and we seek to determine its specificities and roles. The functional consequences of these mutations will be based on the analysis of (1) NO production by fluorimetry with DAF-2DA, (2) cellular morphology by microscopy, and (3) algae responses to abiotic stress

CONCLUSION

Findings

DATA AVAILABILITY STATEMENT