The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K m for the substrate L-Arg of 12 6 5 mM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of L-Arg, suggesting a link between NOS activity and microalgal physiology.

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IntroductionPhotosynthetic organisms respond to nitrogen (N) deprivation with the slowdown of photosynthesis and electron transport resulting in the balance the carbon (C)/N ratio. Under this extreme condition, organisms trigger complex mechanisms to keep growing using different N sources and recycling N containing molecules. In particular, phytoplankton are able to uptake L-arginine (L-Arg) as an organic N source. L-Arg can be assimilated mainly by the arginase, arginine deimidase, arginine decarboxylase or L-amino oxidase pathways.ResultsWe analyzed the effect of different N sources on the growth of the green algae Ostreococcus tauri. N starvation caused an inhibition of culture growth and a decrease in chlorophyll content. The addition of L-Arg to an N-deprived medium promotes a sustained growth rate of O. tauri culture and the increase of chlorophyll levels. The transcript level of genes involved in N uptake and metabolism were increased in N-starved condition while the addition of L-Arg as the sole N source reduced their induction. Since the O. tauri genome lacks the classical pathways to metabolize L-Arg, another enzyme/s may be responsible for L-Arg catabolism. Previously, we characterized the nitric oxide synthase (NOS) enzyme from O. tauri (OtNOS) which oxidizes L-Arg producing nitric oxide (NO) and citrulline. The NOS inhibitor L-NAME blocks the effect promoted by L-Arg on N-deprived O. tauri growth. Besides, NO level increased in O. tauri cells growing in L-Arg containing medium, suggesting the participation of OtNOS enzyme in L-Arg metabolism during N starvation.DiscussionOur hypothesis suggests that, after NOS-dependent Arg degradation, non-enzymatic oxidation of NO produces N oxides (mainly NO2-) that are re-incorporated to the N primary metabolism. As expected, N deprivation increases the lipid content in Ostreococcus. The addition of L-Arg or NO2- as the sole N sources showed a similar increase in lipid content to N deprivation. In summary, our results demonstrate that L-Arg is able to function as N source in Ostreococcus. The evidences on an alternative pathway of N supply and metabolism in a photosynthetic microorganism are discussed. These results could also allow the development of biotechnological tools for increasing lipid production for industry.