All living organisms require constant nitrogen (N) supply for growth and survival. N is an essential macronutrient necessary for the biosynthesis of amino acids, proteins and nucleic acids. The ocean’s N cycle is driven by complex microbial transformations, including N fixation, assimilation, nitrification and denitrification. N can be incorporated in eukaryotic organisms from either organic or inorganic forms. The availability and concentrations of inorganic N sources fluctuate in the environment limiting growth and productivity (Raven et al., 2014). Ammonia (NH₄ ⁺), nitrite (NO₂ ^-) and nitrate (NO₃ ^-) with different spatial distributions in oceans are the most frequent inorganic N sources assimilated by photosynthetic organisms (Gruber and Galloway, 2008).

Based on the function they fulfill within the ecosystem; microalgae possess diverse adaptations for an appropriate N uptake and assimilation. Two adaptive strategies are generally considered when N sources are insufficient: i) increase uptake of N sources by inducing high-affinity transporters and/or by changing growth patterns or chemotaxis, known collectively as N scavenging, and ii) mobilize internal N reserves such as storage proteins and N-rich molecules, known collectively as N salvaging (Andersson et al., 2006; Sanz-Luque et al., 2015; Terrado et al., 2015; Shahar et al., 2020; Lage et al., 2021; van Tol and Armbrust, 2021). The expression of genes that participate in N collecting and recovering is inhibited when the primary N sources are available. This response to N source quality in the growing medium is called N catabolite repression and has been extensively studied in the model system Saccharomyces cerevisiae (Zhang et al., 2018). Assimilation of inorganic N sources requires a significant amount of carbon (C) skeletons and reducing equivalents forcing an increase in photosynthetic products (Foyer et al., 2018). Under N-limitation, many photosynthetic organisms change their C storage pattern in favor of carbohydrates and neutral lipids (NLs) mainly in the form of triacylglycerol (TAG) as a protective response (Illman et al., 2000; Rodolfi et al., 2009; Li et al., 2012; Johnson and Alric, 2013; Rodolfi et al., 2017).

Generally, unicellular algae show little significant ability to use amino acids supplied at near-natural concentrations (Flynn, 1990). In this sense, among 18 amino acids tested, only L-Arg was efficiently assimilated in Chlamydomonas reinhardtii and Volvox carteri (Kirk and Kirk, 1978; Muñoz-Blanco et al., 1990). Munz et al. (2020), recently demonstrated that L-Arg-fed cultures of C. reinhardtii activate N catabolic genes together with N starvation-induced responses such as gametogenesis and TAG accumulation while supporting a growth rate comparable to even higher than NH₄ ⁺-fed cultures. Additionally, L-Arg sustains growth and lipid accumulation in the non-photosynthetic green alga Polytomella parva (Lapina et al., 2022). In photosynthetic eukaryotic organisms, several N-producing ways of L-Arg degradation were described: the arginase pathway which produces ornithine and urea, the arginine decarboxylase (ADC) pathway that converts L-Arg to agmatine and then to N-carbamoyl-putrescine and NH₄ ⁺ (by agmatine iminohydrolase; AIH) and the L-Arg deiminase (ADI) pathway which produces NH₄ ⁺, CO₂, and ATP in 3 step-enzyme reactions (Vallon and Spalding, 2009). Moreover, the L-amino oxidase (LAO) activity can also provide assimilable N from L-Arg (Calatrava et al., 2019).

Picoplankton is the smallest group of microorganisms of the plankton with a diameter of 0.2-3 μm, and are the most abundant primary producers in the oceans, despite representing less than 1% of the photosynthetic biomass (Falkowski, 1994; Field et al., 1998; Falkowski et al., 2008; Seymour, 2014). Therefore, a change in the phytoplankton population will severely affect the biogeochemical cycles. The picoeukaryote Ostreococcus represents a diversified and widely distributed genus within the algae (Le Bihan et al., 2011). Ostreococcus is one of the smallest picoeukaryote, with a very simple cell structure, including only one chloroplast and mitochondrion and no flagella (Six et al., 2005; Cardol et al., 2008; Demir-Hilton et al., 2011). Specifically, in Ostreococcus tauri, we characterized three calcium-dependent protein kinases (CDPKs) and showed that they are involved in the stress response to N deprivation (Caló et al., 2017). Moreover, it was proposed that CDPK signaling originated early in the green lineage is crucial for nutrient perception in Prasinophytes. In a recent study, we described the TOR kinase and its regulation under nutrient stress conditions in O. tauri, highlighting the importance of this microalgae as a model for studying signaling pathways in photosynthetic organisms (Caló et al., 2022).

Additionally, we described a nitric oxide synthase in O. tauri (OtNOS) that metabolizes L-Arg generating NO and citrulline (Foresi et al., 2010; Weisslocker-Schaetzel et al., 2017). The recombinant OtNOS has a Km for the substrate L-Arg of ∼12 μM and its activity is suppressed by the specific inhibitor L-NAME. NOS activity is 3-fold higher during the exponential growth phase than the stationary phase and is increased under high light, evidencing a link between NO production and the microalgal physiology (Foresi et al., 2010).

The influence of different N conditions on O. tauri cell physiology was investigated in the present study. Results showed that N deficiency triggers growth inhibition and a decrease of chlorophyll content in the first days of treatment. On the other hand, L-Arg supplementation to N-deprived cells allows this culture to grow at similar rates to those of N-complete medium for the first 10 days. Based on in silico analysis, we propose a possible pathway of L-Arg catabolism that may overlook the N physiological status. Our results report a study system where N starvation responses are induced without compromising growth, unraveling the way in which cellular N status in eukaryotic phototrophs is sensed and modified. This evidence would allow the development of biotechnological tools that could increase algae growth and lipids content under N deprivation.

In this report, we showed that cultures of O. tauri supplemented with L-Arg display a series of N deficiency responses, including the induction of genes involved in N metabolism and uptake, reduced cellular chlorophyll content and accumulation of lipid storage despite robust growth. Thus, L-Arg-supplemented cultures may trigger the same signaling that induces N starvation. The N starvation responses in L-Arg fed cultures were also observed in C. reinhardtii and P. tricornutum, suggesting a common effect of L-Arg supplementation in alga (Flynn and Wright, 1986; Munz et al., 2020). Triggering N limitation responses requires signaling mechanisms that perceive external and internal N availability. In the model alga C. reinhardtii, transcriptional/proteomic changes upon N starvation have been intensely studied to know the routes of TAG accumulation in the microalgae (Wase et al., 2014). N limitation rapidly induces genes including the classical N catabolite genes for N scavenging and N salvaging, indicating the activation of N remobilization from purines and amino acids while searching for other external N sources (Schmollinger et al., 2014; Park et al., 2015). Additionally, low N resources down-regulate the expression of genes associated with the antenna system, decreasing light absorption and electron transport (Huang et al., 2019).

In this study, we describe that O. tauri can grow at the same time that produces lipids in an N-deficient medium supplemented with L-Arg. Previously, Degraeve-Guilbault et al. (2017); Degraeve-Guilbault et al. (2021) provided a comprehensive study of the glycerolipidome and validated this species as a model for related picoeukaryotes. O. tauri lipids showed unique characteristics that combined traits from green and chromalveolate lineages (Degraeve-Guilbault et al., 2017; Degraeve-Guilbault et al., 2021). Ostreococcus presents a single APC-type transporter (Amino acid/polyamine transporter, NCBI Sequence: XP_003083160.2) orthologous to AOC5/6 (plant cationic amino acid transporters). These transporters facilitate the bidirectional transport of cationic amino acids, thereby supporting critical metabolic functions, such as the synthesis of proteins, NO, and polyamines (Hatzoglou et al., 2004). The fact that O. tauri has an APC-type transporter indicates that this alga will show preferential use of L-Arg over other amino acids (Vallon and Spalding, 2009).

There are several pathways for L-Arg degradation that produce reusable N in photosynthetic organisms ( Figure 7 ). Higher plants use L-Arg as an N source whose utilization is primarily dependent on arginase activity followed by urease that catalyzes the formation of NH₄ ⁺ and CO₂ (Winter et al., 2015). These activities primarily occur during the germination process where a rapid N remobilization from N sources is required for plant growth (Goldraij and Polacco, 2000; Todd et al., 2001; Winter et al., 2015). In addition to germination, arginase plays a key role in the recycling of N during plant senescence and grain production in rice (Ma et al., 2013). However, the arginase enzyme is absent in the O. tauri genome. The evidence supporting the ADI pathway is also weak, as no gene/coding sequence was found. The ADI pathway is present in fungi, algal, and cyanobacterial genomes (Novák et al., 2016; Noens and Lolkema, 2017; Flores et al., 2019; Flores, 2020). For example, Chlorella and Chlamydomonas can utilize L-Arg for growth via the ADI pathway (Laliberté and Hellebust, 1990; Zuo et al., 2012). In addition, Chlamydomonas can also deaminate various amino acids including L-Arg in a N-depleted medium by the action of LAO activity (Calatrava et al., 2019). LAO1 is found in the periplasmic space and was proposed to contribute to N assimilation in oceanic environments. Chlamydomonas genome encodes for a putative intracellular LAO3 (Calatrava et al., 2019). The reaction of LAO enzymes produces ammonium, hydrogen peroxide and the corresponding keto acid; then NH₄ ⁺ can be incorporated and assimilated by algae ( Figure 7 ). In contrast, a homologous sequence coding for this protein is absent in the Ostreococcus genome.

The O. tauri genome presents a coding sequence with partial homology (38% similarity) with ADC from Arabidopsis thaliana, with the two classical domains required for activity (PFAM PS00878 and PS00879). ADC cleaves the carboxyl group of the amino acid group releasing agmatine and CO₂. ADC homologous have been reported in plants and several Chlorella species (Cohen et al., 1983; Beigbeder et al., 1995; Lin and Lin, 2019). Then, agmatine is converted to N-carbamoylputrescine and NH₄ ⁺ by agmatine iminohydrolase (AIH) and then to putrescine by N-carbamoylputrescine amidohydrolase (CPA, Figure 7 ). The agmatine catabolic enzymes, encoded by two AIH1 and AIH2 in Chlamydomonas, are predicted to localize to the mitochondria meaning the ADC pathway would likely be directed to the mitochondria. Both AIH and CPA genes are conserved in Volvox, but not in Ostreococcus (Vallon and Spalding, 2009). Thus, this evidence suggests that there should be another pathway apart from the ones described to get assimilable N from L-Arg in O. tauri.

Our results evince that O. tauri grown in L-Arg produces NO by NOS, which was previously described and characterized in O. tauri (Foresi et al., 2010). NO has a half-life of only a few seconds since it rapidly interacts with O₂, producing nitrogen oxides (NOx), such as NO₃ ^- and NO₂ ^- (Lancaster, 2015). This NO oxidation may be also catalyzed by globins (GLBs), a ubiquitous class of proteins present in all kingdoms (Vinogradov et al., 2006; Figure 7 ). Curiously, we could not find a sequence coding for GLBs in the O. tauri genome. In this sense, our results suggest that NO₂ ^- produced by spontaneous NO oxidation could be assimilated by NIR and incorporated into the N metabolism of the alga. Assimilation of NO₂ ^- derived from NO in flue gas influences the growth of the microalgae Nannochloropsis salina and Phaeodactylun tricornutum (Brown, 1996; Matsumoto et al., 1997). Moreover, Botryococcus braunii can grow in NO₂ ^- as the sole N source for in a range about 2-4 mM while higher concentrations inhibit algae growth (Yang et al., 2004).

So far, this pathway would be a possible way to re-assimilate N from L-Arg in O. tauri. On the other hand, NOS activity produce citrulline, which may be further processed into ornithine and carbamoyl phosphate by the enzyme ornithine carbamoyltransferase (OTC) and then to CO₂ and NH₄ ⁺ by carbamate kinase (CK) again absent in the O. tauri genome (Vallon and Spalding, 2009). The role of NOS in N assimilation from L-Arg is supported by the use of the L-NAME, which prevents growth and NO production in O. tauri grown in L-Arg. Although transgenic lines of O. tauri have been generated using pOtOXLuc transgenics (Degraeve-Guilbault et al., 2020), no commercial mutant lines of O. tauri are currently available. Therefore, employing a pharmacological approach using specific inhibitors is now extremely useful.

Besides N source, the latest data have described NO as a signal molecule in algae (Foresi et al., 2010; Foresi et al., 2017; Astier et al., 2021; Chatelain et al., 2021). NO acts as a signal in the transcriptional and posttranslational regulation of NR and inorganic N transport in Chlamydomonas (Sanz-Luque et al., 2015). NO participates in stress responses, in cell physiology, and is involved in greenhouse gas emission due to the reduction of NO into N₂O in algae (Romano et al., 2011; Kumar et al., 2015; Burlacot et al., 2020). Burlacot et al. (2020) reported that C. reinhardtii produces N₂O in the light by NO reduction catalyzed by flavodiiron proteins (FLVs), whereas in unlighted N₂O production is catalyzed by cytochrome P450 (CYP55). Through a sequence homology search we identify a flavodiiron protein (XP_003075215.2, 60-52% similarity to Chlamydomonas FLVs) and several cytochrome P450 (40-50% similarity to CYP55) in O. tauri. This is extremely interesting for the study of the relationship between NOS function and N₂O production in O. tauri.

This report provides evidence that that directly relates NOS activity with a function in the N metabolism and growth in algae. In a previous work, we analyzed the effect of recombinant OtNOS expressed under IPTG-induced promoter in E. coli, which lacks the gene NOS. The OtNOS expression promotes E. coli growth in a complete nutrient medium and provides a more efficient metabolization of L-Arg as an N source (Correa-Aragunde et al., 2022). Furthermore, the expression of OtNOS under the control of the Cauliflower Mosaic Virus (CaMV) in Nicotiana tabacum enhances growth and seed production, suggesting that OtNOS expression in plants has a straight impact on N metabolism (Nejamkin et al., 2020).

This alga has commercial potential as photoautotrophic cell factories capable of producing high-value biofuels and specialty oils (Degraeve-Guilbault et al., 2017). Advances in bioprocess technology, coupled with strain improvement through traditional methods of mutagenesis or genetic engineering, now offer the potential to improve the economics of oil production. Recently a promising transformation protocol for O. tauri has been reported (Sanchez et al., 2019). The characterization of NOS as an enzyme that metabolizes L-Arg and allows the growth of O. tauri demonstrates a novel and potential biotechnological role of NOS in N metabolism in photosynthetic organisms.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

NF, GM-N, LL, GS and NC-A designed research. GC, FC, AN, NC-A and NF perform the experiments. GM-N, NF and AN analyzed data. NF, NC-A, wrote the paper with the input from the other authors. All authors contributed to the article and approved the submitted version.