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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During evolution, several algae and plants became heterotrophic and lost photosynthesis; however, in most cases, a nonphotosynthetic plastid was maintained. Among these organisms, the colourless alga Polytomella parva is a special case, as its plastid is devoid of any DNA, but is maintained for specific metabolic tasks carried out by nuclear encoded enzymes. This makes P. parva attractive to study molecular events underlying the transition from autotrophic to heterotrophic lifestyle. Here we characterize metabolic adaptation strategies of P. parva in comparison to the closely related photosynthetic alga Chlamydomonas reinhardtii with a focus on the role of plastid‐localized PII signalling protein. Polytomella parva accumulates significantly higher amounts of most TCA cycle intermediates as well as glutamate, aspartate and arginine, the latter being specific for the colourless plastid. Correlating with the altered metabolite status, the carbon/nitrogen sensory PII signalling protein and its regulatory target N‐acetyl‐l‐glutamate‐kinase (NAGK; the controlling enzyme of arginine biosynthesis) show unique features: They have co‐evolved into a stable hetero‐oligomeric complex, irrespective of effector molecules. The PII signalling protein, so far known as a transiently interacting signalling protein, appears as a permanent subunit of the enzyme NAGK. NAGK requires PII to properly sense the feedback inhibitor arginine, and moreover, PII tunes arginine‐inhibition in response to glutamine. No other PII effector molecules interfere, indicating that the PII‐NAGK system in P. parva has lost the ability to estimate the cellular energy and carbon status but has specialized to provide an entirely glutamine‐dependent arginine feedback control, highlighting the evolutionary plasticity of PII signalling system.

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre-gametes mostly used a low-affinity system (LATS) component, but gametes obtained after light treatment of N-deprived pre-gametes expressed both LATS and high-affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light-dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.