The mammalian-type thioredoxin reductase 1 confers a high-light tolerance to the green alga Chlamydomonas reinhardtii.

Reactive oxygen species (ROS) can both act as a poison causing cell death and important signaling molecules among various organisms. Photosynthetic organisms inevitably produce ROS, making the appropriate elimination of ROS an essential strategy for survival. Interestingly, the unicellular green alga Chlamydomonas reinhardtii expresses a mammalian form of thioredoxin reductase, TR1, which functions as a ROS scavenger in animal cells. To investigate the properties of TR1 in C. reinhardtii, we generated TR1 knockout strains using CRISPR/Cas9-based genome editing. We found a reduced tolerance to high-light and ROS stresses in the TR1 knockout strains compared to the parental strain. In addition, the regulation of phototactic orientation, known to be regulated by ROS, was affected in the knockout strains. These results suggest that TR1 contributes to a ROS-scavenging pathway in C. reinhardtii.

Electron transfer via cytochrome b6f complex displays sensitivity to antimycin A upon STT7 kinase activation.

The cytochrome b6f complex (b6f) has been initially considered as the ferredoxin-plastoquinone reductase (FQR) during cyclic electron flow (CEF) with photosystem I that is inhibited by antimycin A (AA). The binding of AA to the b6f Qi-site is aggravated by heme-ci, which challenged the FQR function of b6f during CEF. Alternative models suggest that PROTON GRADIENT REGULATION5 (PGR5) is involved in a b6f-independent, AA-sensitive FQR. Here, we show in Chlamydomonas reinhardtii that the b6f is conditionally inhibited by AA in vivo and that the inhibition did not require PGR5. Instead, activation of the STT7 kinase upon anaerobic treatment induced the AA sensitivity of b6f which was absent from stt7-1. However, a lock in State 2 due to persisting phosphorylation in the phosphatase double mutant pph1;pbcp did not increase AA sensitivity of electron transfer. The latter required a redox poise, supporting the view that state transitions and CEF are not coercively coupled. This suggests that the b6f-interacting kinase is required for structure-function modulation of the Qi-site under CEF favoring conditions. We propose that PGR5 and STT7 independently sustain AA-sensitive FQR activity of the b6f. Accordingly, PGR5-mediated electron injection into an STT7-modulated Qi-site drives a Mitchellian Q cycle in CEF conditions.

PAM: diverse roles in neuroendocrine cells, cardiomyocytes, and green algae.

Our understanding of the ways in which peptides are used for communication in the nervous and endocrine systems began with the identification of oxytocin, vasopressin, and insulin, each of which is stored in electron-dense granules, ready for release in response to an appropriate stimulus. For each of these peptides, entry of its newly synthesized precursor into the ER lumen is followed by transport through the secretory pathway, exposing the precursor to a sequence of environments and enzymes that produce the bioactive products stored in mature granules. A final step in the biosynthesis of many peptides is C-terminal amidation by peptidylglycine α-amidating monooxygenase (PAM), an ascorbate- and copper-dependent membrane enzyme that enters secretory granules along with its soluble substrates. Biochemical and cell biological studies elucidated the highly conserved mechanism for amidated peptide production and raised many questions about PAM trafficking and the effects of PAM on cytoskeletal organization and gene expression. Phylogenetic studies and the discovery of active PAM in the ciliary membranes of Chlamydomonas reinhardtii, a green alga lacking secretory granules, suggested that a PAM-like enzyme was present in the last eukaryotic common ancestor. While the catalytic features of human and C. reinhardtii PAM are strikingly similar, the trafficking of PAM in C. reinhardtii and neuroendocrine cells and secretion of its amidated products differ. A comparison of PAM function in neuroendocrine cells, atrial myocytes, and C. reinhardtii reveals multiple ways in which altered trafficking allows PAM to accomplish different tasks in different species and cell types.

The cryo-EM structure of the chloroplast ClpP complex.

Protein homoeostasis in plastids is strategically regulated by the protein quality control system involving multiple chaperones and proteases, among them the Clp protease. Here, we determined the structure of the chloroplast ClpP complex from Chlamydomonas reinhardtii by cryo-electron microscopy. ClpP contains two heptameric catalytic rings without any symmetry. The top ring contains one ClpR6, three ClpP4 and three ClpP5 subunits while the bottom ring is composed of three ClpP1C subunits and one each of the ClpR1-4 subunits. ClpR3, ClpR4 and ClpT4 subunits connect the two rings and stabilize the complex. The chloroplast Cpn11/20/23 co-chaperonin, a co-factor of Cpn60, forms a cap on the top of ClpP by protruding mobile loops into hydrophobic clefts at the surface of the top ring. The co-chaperonin repressed ClpP proteolytic activity in vitro. By regulating Cpn60 chaperone and ClpP protease activity, the co-chaperonin may play a role in coordinating protein folding and degradation in the chloroplast.

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas.

Chlamydomonas reinhardtii can grow photosynthetically using CO2 or in the dark using acetate as the carbon source. In the light in air, the CO2 concentrating mechanism (CCM) of C. reinhardtii accumulates CO2, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO2 concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO2 (0.04% CO2 [v/v] in air). These strains were not able to grow at very low CO2 (<0.02% CO2 [v/v] in air), and their ability to accumulate inorganic carbon (Ci = CO2 + HCO3-) was reduced. At low CO2 concentrations, the CCM is needed to both deliver Ci to Rubisco and to minimize the leak of CO2 generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO2 released from respiration and photorespiration as well as the CO2 leaked from the chloroplast to HCO3- thus "recapturing" this potentially lost CO2.

The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

Subcellular Localizations of Catalase and Exogenously Added Fatty Acid in Chlamydomonas reinhardtii.

Fatty acids are important biological components, yet the metabolism of fatty acids in microalgae is not clearly understood. Previous studies found that Chlamydomonas reinhardtii, the model microalga, incorporates exogenously added fatty acids but metabolizes them differently from animals and yeast. Furthermore, a recent metabolic flux analysis found that the majority of lipid turnover in C. reinhardtii is the recycling of acyl chains from and to membranes, rather than ß -oxidation. This indicates that for the alga, the maintenance of existing acyl chains may be more valuable than their breakdown for energy. To gain cell-biological knowledge of fatty acid metabolism in C. reinhardtii, we conducted microscopy analysis with fluorescent probes. First, we found that CAT1 (catalase isoform 1) is in the peroxisomes while CAT2 (catalase isoform 2) is localized in the endoplasmic reticulum, indicating the alga is capable of detoxifying hydrogen peroxide that would be produced during ß-oxidation in the peroxisomes. Second, we compared the localization of exogenously added FL-C16 (fluorescently labelled palmitic acid) with fluorescently marked endosomes, mitochondria, peroxisomes, lysosomes, and lipid droplets. We found that exogenously added FL-C16 are incorporated and compartmentalized via a non-endocytic route within 10 min. However, the fluorescence signals from FL-C16 did not colocalize with any marked organelles, including peroxisomes. During triacylglycerol accumulation, the fluorescence signals from FL-C16 were localized in lipid droplets. These results support the idea that membrane turnover is favored over ß-oxidation in C. reinhardtii. The knowledge gained in these analyses would aid further studies of the fatty acid metabolism.

Plant type I metacaspases are proteolytically active proteases despite their hydrophobic nature.

Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta.

Involvement of Carbonic Anhydrase CAH3 in the Structural and Functional Stabilization of the Water-Oxidizing Complex of Photosystem II from Chlamydomonas reinhardtii.

The involvement of carbonic anhydrases (CA) and CA activity in the functioning of photosystem II (PSII) has been studied for a long time and has been shown in many works. However, so far only for CAH3 from Chlamydomonas reinhardtii there is evidence for its association with the donor side of PSII, where the CA activity of CAH3 can influence the functioning of the water-oxidizing complex (WOC). Our results suggest that CAH3 is also involved in the organization of the native structure of WOC independently of its CA activity. It was shown that in PSII preparations from wild type (WT) the high O2-evolving activity of WOC was observed up to 100 mM NaCl in the medium and practically did not decrease with increasing incubation time with NaCl. At the same time, the WOC function in PSII preparations from CAH3-deficient mutant cia3 is significantly inhibited already at NaCl concentrations above 35 mM, reaching 50% at 100 mM NaCl and increased incubation time. It is suggested that the absence of CAH3 in PSII from cia3 causes disruption of the native structure of WOC, allowing more pronounced conformational changes of its proteins and, consequently, suppression of the WOC active center function, when the ionic strength of the medium is increased. The results of Western blot analysis indicate a more difficult removal of PsbP protein from PSII of cia3 at higher NaCl concentrations, apparently due to the changes in the intermolecular interactions between proteins of WOC in the absence of CAH3. At the same time, the values of the maximum quantum yield of PSII did not practically differ between preparations from WT and cia3, indicating no effect of CAH3 on the photoinduced electron transfer in the reaction center of PSII. The obtained results indicate the involvement of the CAH3 protein in the native organization of the WOC and, as a consequence, in the stabilization of its functional state in PSII from C. reinhardtii.

Bi-directional electron transfer between H2 and NADPH mitigates light fluctuation responses in green algae.

The metabolism of green algae has been the focus of much research over the last century. These photosynthetic organisms can thrive under various conditions and adapt quickly to changing environments by concomitant usage of several metabolic apparatuses. The main electron coordinator in their chloroplasts, nicotinamide adenine dinucleotide phosphate (NADPH), participates in many enzymatic activities and is also responsible for inter-organellar communication. Under anaerobic conditions, green algae also accumulate molecular hydrogen (H2), a promising alternative for fossil fuels. However, to scale-up its accumulation, a firm understanding of its integration in the photosynthetic apparatus is still required. While it is generally accepted that NADPH metabolism correlates to H2 accumulation, the mechanism of this collaboration is still vague and relies on indirect measurements. Here, we investigated this connection in Chlamydomonas reinhardtii using simultaneous measurements of both dissolved gases concentration, NADPH fluorescence and electrochromic shifts at 520-546 nm. Our results indicate that energy transfer between H2 and NADPH is bi-directional and crucial for the maintenance of redox balance under light fluctuations. At light onset, NADPH consumption initially eventuates in H2 evolution, which initiates the photosynthetic electron flow. Later on, as illumination continues the majority of NADPH is diverted to the Calvin-Benson-Bassham cycle. Dark onset triggers re-assimilation of H2, which produces NADPH and so, enables initiation of dark fermentative metabolism.

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase.

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H2 oxidation activity, [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH.

The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of control by epistasy of synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feedback on the initiation of LSU translation.

Bardet-Biedl syndrome 3 protein promotes ciliary exit of the signaling protein phospholipase D via the BBSome.

Certain ciliary signaling proteins couple with the BBSome, a conserved complex of Bardet-Biedl syndrome (BBS) proteins, to load onto retrograde intraflagellar transport (IFT) trains for their removal out of cilia in Chlamydomonas reinhardtii. Here, we show that loss of the Arf-like 6 (ARL6) GTPase BBS3 causes the signaling protein phospholipase D (PLD) to accumulate in cilia. Upon targeting to the basal body, BBSomes enter and cycle through cilia via IFT, while BBS3 in a GTP-bound state separates from BBSomes, associates with the membrane, and translocates from the basal body to cilia by diffusion. Upon arriving at the ciliary tip, GTP-bound BBS3 binds and recruits BBSomes to the ciliary membrane for interacting with PLD, thus making the PLD-laden BBSomes available to load onto retrograde IFT trains for ciliary exit. Therefore, BBS3 promotes PLD exit from cilia via the BBSome, providing a regulatory mechanism for ciliary signaling protein removal out of cilia.

Multiple putative methemoglobin reductases in C. reinhardtii may support enzymatic functions for its multiple hemoglobins.

The ubiquitous nature of hemoglobins, their presence in multiple forms and low cellular expression in organisms suggests alternative physiological functions of hemoglobins in addition to oxygen transport and storage. Previous research has proposed enzymatic function of hemoglobins such as nitric oxide dioxygenase, nitrite reductase and hydroxylamine reductase. In all these enzymatic functions, active ferrous form of hemoglobin is converted to ferric form and reconversion of ferric to ferrous through reduction partners is under active investigation. The model alga C. reinhardtii contains multiple globins and is thus expected to have multiple putative methemoglobin reductases to augment the physiological functions of the novel hemoglobins. In this regard, three putative methemoglobin reductases and three algal hemoglobins were characterized. Our results signify that the identified putative methemoglobin reductases can reduce algal methemoglobins in a nonspecific manner under in vitro conditions. Enzyme kinetics of two putative methemoglobin reductases with methemoglobins as substrates and in silico analysis support interaction between the hemoglobins and the two reduction partners as also observed in vitro. Our investigation on algal methemoglobin reductases underpins the valuable chemistry of nitric oxide with the newly discovered hemoglobins to ensure their physiological relevance, with multiple hemoglobins probably necessitating the presence of multiple reductases.

Evolutive differentiation between alga- and plant-type plastid terminal oxidase: Study of plastid terminal oxidase PTOX isoforms in Marchantia polymorpha.

The liverwort Marchantia polymorpha contains two isoforms of the plastid terminal oxidase (PTOX), an enzyme that catalyzes the reduction of oxygen to water using plastoquinol as substrate. Phylogenetic analyses showed that one isoform, here called MpPTOXa, is closely related to isoforms occurring in plants and some algae, while the other isoform, here called MpPTOXb, is closely related to the two isoforms occurring in Chlamydomonas reinhardtii. Mutants of each isoform were created in Marchantia polymorpha using CRISPR/Cas9 technology. While no obvious phenotype was found for these mutants, chlorophyll fluorescence analyses demonstrated that the plastoquinone pool was in a higher reduction state in both mutants. This was visible at the level of fluorescence measured in dark-adapted material and by post illumination fluorescence rise. These results suggest that both isoforms have a redundant function. However, when P700 oxidation and re-reduction was studied, differences between these two isoforms were observed. Furthermore, the mutant affected in MpPTOXb showed a slight alteration in the pigment composition, a higher non-photochemical quenching and a slightly lower electron transport rate through photosystem II. These differences may be explained either by differences in the enzymatic activities or by different activities attributed to preferential involvement of the two PTOX isoforms to either linear or cyclic electron flow.

The BBSome restricts entry of tagged carbonic anhydrase 6 into the cis-flagellum of Chlamydomonas reinhardtii.

The two flagella of Chlamydomonas reinhardtii are of the same size and structure but display functional differences, which are critical for flagellar steering movements. However, biochemical differences between the two flagella have not been identified. Here, we show that fluorescence protein-tagged carbonic anhydrase 6 (CAH6-mNG) preferentially localizes to the trans-flagellum, which is organized by the older of the two flagella-bearing basal bodies. The uneven distribution of CAH6-mNG is established early during flagellar assembly and restored after photobleaching, suggesting that it is based on preferred entry or retention of CAH6-mNG in the trans-flagellum. Since CAH6-mNG moves mostly by diffusion, a role of intraflagellar transport (IFT) in establishing its asymmetric distribution is unlikely. Interestingly, CAH6-mNG is present in both flagella of the non-phototactic bardet-biedl syndrome 1 (bbs1) mutant revealing that the BBSome is involved in establishing CAH6-mNG flagellar asymmetry. Using dikaryon rescue experiments, we show that the de novo assembly of CAH6-mNG in flagella is considerably faster than the removal of ectopic CAH6-mNG from bbs flagella. Thus, different rates of flagellar entry of CAH6-mNG rather than its export from flagella is the likely basis for its asymmetric distribution. The data identify a novel role for the C. reinhardtii BBSome in preventing the entry of CAH6-mNG specifically into the cis-flagellum.

High-Resolution Crystal Structure of Chloroplastic Ribose-5-Phosphate Isomerase from Chlamydomonas reinhardtii-An Enzyme Involved in the Photosynthetic Calvin-Benson Cycle.

The Calvin-Benson cycle is the key metabolic pathway of photosynthesis responsible for carbon fixation and relies on eleven conserved enzymes. Ribose-5-phosphate isomerase (RPI) isomerizes ribose-5-phosphate into ribulose-5-phosphate and contributes to the regeneration of the Rubisco substrate. Plant RPI is the target of diverse post-translational modifications including phosphorylation and thiol-based modifications to presumably adjust its activity to the photosynthetic electron flow. Here, we describe the first experimental structure of a photosynthetic RPI at 1.4 Å resolution. Our structure confirms the composition of the catalytic pocket of the enzyme. We describe the homo-dimeric state of the protein that we observed in the crystal and in solution. We also map the positions of previously reported post-translational modifications and propose mechanisms by which they may impact the catalytic parameters. The structural data will inform the biochemical modeling of photosynthesis.

[Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii].

In a mixotrophic Chlamydomonas reinhardtii culture, the expression levels of genes encoding primary metabolic enzymes and chloroplast plastid transporters were analyzed. For the majority of the genes studied, their expression levels decreased as they approached the final stages of culture growth. During the period of exponential growth, the expression profiles changed more intensively than during the stationary phase. In the middle of exponential growth, significant changes of mRNA profiles reflected reorganization of metabolism, with an emphasis on the induction of lipid synthesis, accompanied by alterations in carbon fluxes through biochemical pathways and a shift in the energy balance between the plastid and cytosol.

Ascorbate peroxidase 4 plays a role in the tolerance of Chlamydomonas reinhardtii to photo-oxidative stress.

Ascorbate peroxidase (APX; EC 1.11.1.11) activity and transcript levels of CrAPX1, CrAPX2, and CrAPX4 of Chlamydomonas reinhardtii increased under 1,400 µE·m-2·s-1 condition (HL). CrAPX4 expression was the most significant. So, CrAPX4 was downregulated using amiRNA technology to examine the role of APX for HL acclimation. The CrAPX4 knockdown amiRNA lines showed low APX activity and CrAPX4 transcript level without a change in CrAPX1 and CrAPX2 transcript levels, and monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) activities and transcript levels. Upon exposure to HL, CrAPX4 knockdown amiRNA lines appeared a modification in the expression of genes encoding the enzymes in the ascorbate-glutathione cycle, including an increase in transcript level of CrVTC2, a key enzyme for ascorbate (AsA) biosynthesis but a decrease in MDAR and DHAR transcription and activity after 1 h, followed by increases in reactive oxygen species production and lipid peroxidation after 6 h and exhibited cell death after 9 h. Besides, AsA content and AsA/DHA (dehydroascorbate) ratio decreased in CrAPX4 knockdown amiRNA lines after prolonged HL treatment. Thus, CrAPX4 induction together with its association with the modulation of MDAR and DHAR expression for AsA regeneration is critical for Chlamydomonas to cope with photo-oxidative stress.

TOR kinase activity in Chlamydomonas reinhardtii is modulated by cellular metabolic states.

Target of rapamycin (TOR) kinase is a sensor and a central integrator of internal and external metabolic cues. However, in algae and in higher plants, the components of TOR kinase signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in Chlamydomonas reinhardtii using the phosphorylation status of its putative downstream target, CrS6K. Using this assay, we probe the modulation of cellular TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy, and nitrogen (N) starvation. Importantly, we uncover that excess acetate in the medium leads to high cellular reactive oxygen species levels, triggering autophagy and a concomitant drop in TOR kinase activity in a dose-dependent manner, thus leading to a N-starvation-like cellular phenotype, even when nitrogen is present.