N-Acetyl-L-glutamate Kinase of Chlamydomonas reinhardtii: In Vivo Regulation by PII Protein and Beyond.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the rate-limiting step in the ornithine/arginine biosynthesis pathway in eukaryotic and bacterial oxygenic phototrophs. NAGK is the most highly conserved target of the PII signal transduction protein in Cyanobacteria and Archaeplastida (red algae and Chlorophyta). However, there is still much to be learned about how NAGK is regulated in vivo. The use of unicellular green alga Chlamydomonas reinhardtii as a model system has already been instrumental in identifying several key regulation mechanisms that control nitrogen (N) metabolism. With a combination of molecular-genetic and biochemical approaches, we show the existence of the complex CrNAGK control at the transcriptional level, which is dependent on N source and N availability. In growing cells, CrNAGK requires CrPII to properly sense the feedback inhibitor arginine. Moreover, we provide primary evidence that CrPII is only partly responsible for regulating CrNAGK activity to adapt to changing nutritional conditions. Collectively, our results suggest that in vivo CrNAGK is tuned at the transcriptional and post-translational levels, and CrPII and additional as yet unknown factor(s) are integral parts of this regulation.

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

Nitric oxide (NO) acts as a key signaling molecule in higher plants, regulating many physiological processes. Several photosynthetic algae from different lineages are also known to produce NO. However, it remains unclear whether this messenger is produced by non-photosynthetic algae. Among these organisms, the colorless alga Polytomella parva is a special case, as it has lost not only its plastid genome, but also nitrate reductase and nitrite reductase. Up to now, the question of whether NO synthesis occurs in the absence of functional nitrate reductase (NR) and the assimilation of nitrates/nitrites in P. parva has not been elucidated. Using spectrofluorometric assays and confocal microscopy with NO-sensitive fluorescence dye, we demonstrate L-arginine-dependent NO synthesis by P. parva cells. Based on a pharmacological approach, we propose the existence of arginine-dependent NO synthase-like activity in this non-photosynthetic alga. GC-MS analysis provides primary evidence that P. parva synthesizes putrescine, which is not an NO source in this alga. Moreover, the generated NO causes the S-nitrosation of protein cysteine thiol groups. Together, our data argue for NR-independent NO synthesis and its active role in S-nitrosation as an essential post-translational modification in P. parva.