Symbiotic interactions in the lichen Ramalina farinacea dramatically modify NO biosynthetic source in Trebouxia microalgae

NO is a multifaceted molecule, key in functions such as abiotic stress tolerance and symbioses establishment and permanence. Lichens are complex symbiotic associations of microalgae, fungi and prokaryotes that release NO under stress conditions such as dessication-rehydration cycles and the presence of xenobiotics. NO synthase (NOS) oxidises l-arginine to produce NO in animals and some aquatic microalgae, while nitrate reductase (NR) reduces nitrate to NO in plants and fungi. Inhibition studies suggest that both activities might be present in thalli. Due to its multipartner compotition, our hypothesis is that Ramalina farinacea biosynthesises NO through both oxidative (NOS) and reductive (NR) enzymatic pathways. NR activity was quantified with a method optimised for lichens using NADH or/and NADPH, and NOS with a commercial kit in R. farinacea thalli and cultures of the isolated main symbionts: R. farinacea mycobiont, and Trebouxia jamesii and Trebouxia lynnae phycobionts. Inhibition studies in vitro were performed with L-NAME and tungstate. Immunodetection was carried out with specific polyclonal antibodies (anti-plant NADH-NR and anti-iNOS animal isoform). NADH-NR specific activity of R. farinacea is an order of magnitude higher than Arabidopsis thaliana's and in the range of the chlorophyte Ulva intestinalis. R. farinacea mycobiont possesses a canonical plant-like Moco-NR, while Trebouxia phycobionts' NR activity presents interesting peculiarities. NOS has not been immunodetected and NOS-like activity is inhibited by L-NAME only partially in T. jamesii. Despite NOS-like activity is very high in the isolated microalgae and fungus, it is strongly depressed in the holobiont. In summary, NR activity seems to be the main source of NO biosynthesis for the holobiont R. farinacea but it presents intriguing features that deserve further study.