Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

PII superfamily consists of widespread signal transduction proteins found in all domains of life. Whereas they are well-studied in Archaea, Bacteria and Chloroplastida, no PII homolog has been analyzed in Rhodophyta (red algae), where PII is encoded by a chloroplast localized glnB gene. Here, we characterized relevant sensory properties of PII from the red alga Porphyra purpurea (PpPII) in comparison to PII proteins from different phyla of oxygenic phototrophs (cyanobacteria, Chlamydomonas and Physcomitrella) to assess evolutionary conservation versus adaptive properties. Like its cyanobacterial counterparts, PpPII binds ATP/ADP and 2-oxoglutarate in synergy with ATP. However, green algae and land plant PII proteins lost the ability to bind ADP. In contrast to PII proteins from green algae and land plants, PpPII enhances the activity of N-acetyl-L-glutamate kinase (NAGK) and relieves it from feedback inhibition by arginine in a glutamine-independent manner. Like PII from Chloroplastida, PpPII is not able to interact with the cyanobacterial transcriptional co-activator PipX. These data emphasize the conserved role of NAGK as a major PII-interactor throughout the evolution of oxygenic phototrophs, and confirms the specific role of PipX for cyanobacteria. Our results highlight the PII signaling system in red algae as an evolutionary intermediate between Cyanobacteria and Chlorophyta.The PII superfamily were originally described as widely distributed members of a family of cell signaling proteins occurring in all domains of life 1-3 with representatives in almost all bacteria and in nitrogen-fixing archaea 4,5 as well as in oxygenic eukaryotic phototrophs 6 . The canonical PII proteins are the master regulator of nitrogen metabolism and they are encoded by glnB and glnK genes 2,7 . The superfamily of PII-like proteins was enlarged by including members that are characterized by the typical structural architecture of PII proteins but lack the typical PROSITE signature pattern of initially characterized PII proteins 7 . Those PII homologues, which contain the typically conserved PROSITE motifs of GlnB/GlnK-like PII proteins are referred as canonical PII proteins.The canonical PII proteins have fundamentals roles as energy/carbon/nitrogen sensors 8 . The binding of small effector molecules to PII proteins allows modulation of different cellular functions. Competitive binding of ATP or ADP and synergistic binding of 2-oxoglutarate (2-OG) with ATP enables PII to estimate the current energy and nitrogen/carbon status of the cells. The various effector molecule binding events cause signaling through conformational changes within the PII tirmer, which in turn allows PII to bind to different interacting partners to regulate the actual metabolic situation. Under conditions of high 2-OG levels (poor nitrogen supply), the ATP-dependent binding of 2-OG to PII causes strong conformational changes in the T-loop, which in turn impairs the interaction of PII proteins with different targets 7 . In all examined cases studied so far, PI...

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Chemotactic behavior of Chlamydomonas reinhardtii is altered during the sexual life cycle. Unlike vegetative cells and noncompetent pregametes, mature gametes did not show chemotaxis to ammonium. Loss of chemotaxis to ammonium in mating-competent cells is controlled by gamete-specific genes that are common for both mating-type gametes. Change of chemotaxis mode requires the sequential action of the two environmental signals: removal of ammonium from the medium and light. The mutants lrg1, lrg3, and lrg4 affected in the light-dependent step of sexual differentiation exhibited the loss of chemotaxis to ammonium in the absence of light. These data indicate that there are common components in the signaling pathways that control change of chemotactic behavior and forming of mating competence in gametes.

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