Introduction

Nitric oxide (NO), initially discovered in 1772 by the English scientist Joseph Priestley and referred to as "nitrous air" due to its combustive properties, underwent a significant paradigm shift in biological significance during the 1980s, when the acetylcholine-induced smooth muscle relaxation was shown to be dependent on endothelial cells (Priestley 1776; Chataigneau et al. 1999). It was found that endothelial cells release a chemical signal (endothelium-derived relaxation factor, EDRF) which appeared to be very labile. Further experiments revealed that EDRF is no other than the gaseous free radical, NO (Furchgott et al. 1987; Ignarro et al. 1987). Subsequently, research revealed NO as a crucial cytotoxic agent in the immune system and signaling molecule in the nervous system (Chachlaki and Prevot 2020). Simultaneously, these advancements coincided with the identification of three key isozymes regulating NO synthesis in mammals: neuronal nitric oxide synthase (nNOS or NOS-I), inducible nitric oxide synthase (iNOS or NOS-II), and endothelial nitric oxide synthase (eNOS or NOS-III). Mammalian NO synthases (mNOSs) are highly regulated, complex enzymes responsible for catalyzing the oxidation of L-arginine to produce NO and L-citrulline (Fukuto and Wink 2018; Griffith and Stuehr 1995; Stuehr et al. 2004). Subsequent studies on NOS have revealed NO’s extensive involvement in vital processes in higher organisms, including vascular tone control, blood pressure regulation, immune defense against pathogens, cancer, hormonal balance, nerve cell transmission, and the promotion of angiogenesis (Alderton et al. 2001; Förstermann and Sessa 2012). The groundbreaking discoveries of the 1990s served as a catalyst, propelling an intensified focus on NO research, extending its purview beyond animals to encompass plant studies. The initial investigations focused on NO as an airborne pollutant interacting with above-ground plant structures (Wellburn et al. 1972; Oksanen and Soppela 2021). However, NO underwent a perceptual metamorphosis when plant NO emissions were linked to nitrate reductase (NR) activity, establishing NO as a key regulator of plant physiological processes (Yamasaki 2000). In the early phase, investigations were confined to legume species (Glycine spp., Psophocarpus tetragonolobus, Neonotonia wightii, Pueraria spp.), known for their distinct nitrogen metabolism. However, a subsequent phase of plant NO research, commencing in 1996, featured a more diversified selection of experimental plant species, e.g., soybean, lupine, potato, flowers, and fruits (Millar and Day 1996; Cueto et al. 1996; Noritake et al. 1996; Leshem and Haramaty 1996).

This expanded exploration illuminated the multifaceted role of NO in various dimensions of plant growth and development (Šírová et al. 2011). During seed germination, NO-mediated suppression of cytochrome oxidase (COX) enables fine-tuning of respiration to prevent tissues from becoming anoxic, while in pollen tube growth, NO acts as a regulator, influencing orientation through cGMP signaling (Prado et al. 2008 Over the preceding two decades, NO has emerged as a pivotal chemical messenger, playing a central role in modulating the synthesis of essential plant hormones, including abscisic acid, indole-3-acetic acid, salicylic acid, ethylene, and cytokinin. The intricate interplay of NO with these hormones results in complex signaling cascades, impacting transcriptome and proteome levels across diverse plant species (Hussain et al. 2016). Additionally, numerous studies have presented compelling evidence demonstrating interactions between NO and key signaling entities, such as melatonin, hydrogen sulfide, and reactive oxygen species (ROS), playing a crucial role in modulating various plant functions, particularly under stressful conditions (Khan et al. 2023).

Furthermore, NO production encompasses both oxidative pathways, such as L-arginine or hydroxylamine-based NO synthesis, and reductive pathways, exemplified by nitrate reduction (Gupta Kapuganti et al. 2010). However, the L-arginine-dependent oxidative NO production in plants has been a subject of extensive discourse and scrutiny in recent years within the scientific community (Yamasaki 2000). Historically, two candidate proteins, a variant of the glycine–decarboxylase-complex-protein P and AtNOS1 from Arabidopsis thaliana, were initially proposed for NOS activity in higher plants. However, subsequent investigations disproved these proposals, introducing additional complexity to the quest for a definitive answer. AtNOS, initially considered a potential NOS, was identified as a GTPase and subsequently renamed AtNOA (Chandok et al. 2003). In a milestone publication, a plant NOS was identified and characterized in the green algae Ostreococcus tauri. The OtNOS sequence demonstrates a 44% resemblance to human endothelial NOS and 45% to inducible NOS and neuronal NOS, showcasing a noteworthy conservation across all domains responsible for cofactor binding and dimerization. This groundbreaking discovery, dating back to 2010, elicited widespread excitement, marking the identification of NOS homologs in photosynthetic organisms (Foresi et al. 2010). The exploration extended to other species within this taxon, with NOSs being detected in Ostreococcus lucimarinus and Bathycoccus prasinos. Likewise, a multitude of sequences were identified in algae, specifically in Chlorophytes and Streptophytes (Jeandroz et al. 2016; Kumar et al. 2015; Nejamkin et al. 2020 et al. 2022; Santolini et al. 2017), as well as in diatoms and dinoflagellates (Di Dato et al. 2015; Santolini et al. 2017). A notable discovery in this realm was the identification of a mammalian-like NOS, named syNOS, in the photosynthetic diazotroph Synechococcus sp. PCC 7335 (Fig. 1). SyNOS, the first photosynthetic prokaryotic NOS with a mammalian NOSred homolog, exhibited an unusual globin domain (NOSg) N-terminal to NOSox. Subsequently, recent years have witnessed the identification of NOSs in certain cyanobacteria (Correa-Aragunde et al. 2018; Moroz et al. 2020; Gupta and Mishra 2022), uncovering a greater diversity of structures in these prokaryotes, further expanding our understanding of the evolution and functional diversity of NOS in photosynthetic organisms. Additionally, this revelation prompted thought-provoking inquiries about the roles that NOSs play in diverse photosynthetic organisms distinct from animals. This review offers a comprehensive exploration of crucial aspects, providing insights into recent developments concerning NOS in photosynthetic organisms. It sheds light on the structural diversity observed in prokaryotic NOS sequences identified in cyanobacteria, in addition to delving into the biological functions of NOSs in photosynthetic organisms.

Fig. 1
figure 1

Significant milestones in NOS-derived NO research: The timeline of key discoveries in NO research spans several decades and highlights pivotal moments in understanding the role of NO in various physiological processes. In the 1930s, nitrogen oxides (NOx), including NO, were identified as major contributors to air pollution, associated with smog formation and respiratory issues in urban areas. The year 1987 marked a significant milestone with the publication of "Endothelium-Derived Relaxing Factor (EDRF): NO by Ignarro, Byrns, and Wood. In 1991, researchers achieved a breakthrough by isolating and characterizing the enzyme responsible for NO production, named NOS. This discovery paved the way for NO to be named the ‘‘Molecule of the Year’’ in 1992 by the journal Science. The remarkable contributions of Furchgott, Ignarro, and Murad led to the Nobel Prize in Physiology or Medicine in 1998 for their work on NO as a signaling molecule. Subsequent years witnessed the characterization of the first NOS from green algae in 2010 and a significant milestone in 2018 with the characterization of the first NOS from cyanobacteria, providing further insights into the evolution and diverse roles of NOS across different organisms

Full size image

Tracing the Origins of Nitric Oxide and Nitric Oxide Synthase

The evolutionary trajectory of NOS is a captivating narrative that spans the epochs of Earth’s history. Around 4 billion years ago in the Hadean eon, NO emerged from Earth’s early atmosphere, a product of dynamic interactions involving dinitrogen, carbon dioxide, and water vapor (Chyba and Sagan 1991; Nisbet and Sleep 2001). With the advent of life during the Archean eon, unicellular microbes harnessed ammonium ions (NH4+) as a vital nitrogen source, incorporating them into essential biological molecules (Schopf 2006). As microbial life evolved, the utilization of nitrate (NO3) and nitrite (NO2) as nitrogen alternatives became prevalent when NH4+ availability declined. The microbial process of denitrification emerged as a pivotal mechanism, efficiently converting NO3 to N2, contributing to energy metabolism (Rothschild et al. 2003). The subsequent Great Oxygenation Event, facilitated by cyanobacteria, ushered in elevated oxygen levels but disrupted both abiotic NO synthesis and biological denitrification (Planavsky et al. 2014). Minerals such as iron acted as shields against oxygen toxicity, providing a conducive environment for the evolution of antioxidant capabilities (Feelisch and Martin 1995). In this intricate evolutionary tapestry, NOSs emerged as a defense mechanism, particularly in prokaryotes (Rőszer and Rőszer 2012). Existing in diverse microbial species, including cyanobacteria, NOSs utilize L-arginine to generate L-citrulline and NO, forming a protective shield against the deleterious effects of oxygen. This generated NO acts as a safeguard, directly interacting with oxygen radicals and ozone or indirectly preventing oxidation in vulnerable biomolecules (Feelisch and Martin 1995). Additionally, the oxidized derivatives of NO, specifically NO3 and NO2, contribute positively to cellular adaptation to oxygen by modulating cellular respiration (Chaudhari et al. 2017). Furthermore, numerous systematic and detailed evolutionary studies have provided initial clues about the origin and diversity of NOS. However, there is still much to unravel in this evolutionary narrative (Gusarov et al. 2009 2008; Sudhamsu and Crane 2009). To address this, we executed a phylogenetic analysis encompassing 23 NOS sequences extracted from diverse organisms (Fig. 2). Supplementary file S1 presents a detailed methodology employed for the phylogenetic analysis. The evolution of NOS sequences demonstrates significant divergence across different kingdoms of life. A notable example is the contrast between bacterial and eukaryotic NOS proteins. Bacterial NOS, found in prokaryotes, exhibits structural and functional disparities compared to its eukaryotic counterparts. This divergence is not only a testament to the vast evolutionary timescales but also underscores the versatility of NOS in fulfilling diverse biological functions. Within a species, the divergent evolution of NOS genes is a dynamic process shaped by selective pressures. For instance, neuronal NOS and endothelial NOS in mammals showcase divergent evolution, contributing to the fine-tuning of NO production for specialized cellular functions. This divergence within the NOS gene family highlights how gene duplications have allowed for the specialization of functions, enabling each isoform to contribute uniquely to physiological processes. Paradoxically, convergent evolution in NOS proteins manifests as functional similarities across diverse organisms despite genomic distinctions. The selective pressures favoring NO production as a crucial signaling molecule contribute to this convergence. For instance, bacterial NOS-like enzymes in Bacillus subtilis, algal NOS in Ostreococcus tauri, and cyanobacterial NOS in Synechococcus sp. PCC 7335 exemplify this phenomenon. The shared functionality emphasizes the fundamental role of NO in cellular signaling pathways, transcending taxonomic boundaries. In a previous study, the comparison of the NOS gene-specific tree with the species-specific tree, derived from 16S ribosomal RNA sequences, revealed noteworthy differences in tree topology. Author suggested a distinctive evolution of NOS, deviating from the linear evolution of species (Sudhamsu and Crane 2009). The tantalizing proposition of horizontal gene transfer events shaping NOS evolution among species adds an exciting layer to how NOS evolves. It is like discovering a new thread in the complex pattern of how genes adapt and diversify over time.

Fig. 2
figure 2

Phylogenetic relationship of NOS homologs in the Tree of Life—representative of the bacteria, cyanobacteria, algae, diatom, dinoflagellate, slime mold, and mammal phyla

Full size image

Evolutionary Symphony of Cyanobacterial Nitric Oxide Synthases

To further elucidate the structural heterogeneity and distribution patterns of NOS within cyanobacteria, a comprehensive in-silico homology search was conducted using Basic Local Alignment Search Tool (BLAST). Supplementary file S2 presents a detailed methodology employed for the phylogenetic analysis. Phylogeny of NOS homologs displayed six different clusters (Fig. 3) with strongly supported bootstrap values. The analysis of cyanobacterial NOS protein domains using the NCBI conserved domain database revealed that clusters I is represented by NOS-like proteins, which consist only of putative catalytic evolutionary conserved NOSoxy domain. This architecture is typical of bacterial NOS and is found exclusively in the heterocytous strains of Nostocales (Nejamkin et al. 2023). The presence of the NOSoxy domain, with conserved residues essential for catalysis, suggests it to be the core enzyme for NOS-dependent NO synthesis. Notably, this observation prompts that the multidomain architecture of NOS in eukaryotes may have evolved from a single NOS oxygenase, a feature harbored by prokaryotes, including certain members of Nostocales. Cluster II, constituted by Oscillatoriales and Nostocales, features NOSs which contain a reverse order of the canonical domains, Rossmann-fold binding (NADB) domain, an EF-hand (EFh) Ca2+-binding motif, NAD(P)H-nitrite reductase domain, bacterioferritin-associated ferredoxin (BFD)-like [2Fe-2S]-binding domain, and Ferredoxin-NADP reductase domain (Fig. 3). The presence of this distinctive multidomain architecture in Nostocales and Oscillatoriales suggests potential benefits for the physiological robustness and life strategies of filamentous cyanobacterial strains. These accessory domains likely contribute to diverse functional capacities, enhancing the adaptability and resilience of these cyanobacterial strains in various environmental cues (Crane et al. 1998). In the Cluster III subgroup, homologs closely resembling those in Cluster II are evident. However, it lacks the NAD(P)H-nitrite reductase domain and features Ferredoxin reductase (FNR) instead of Ferredoxin-NADP reductase. Additionally, within the Cluster III subgroup, the absence of the Rossmann-fold binding (NADB) domain is notable only in Scytonema sp. UIC 10036. Cluster IV subgroup comprises homologs to the first described and characterized NOS from Synechococcus sp PCC 7335 (SyNOS) which contains the unusual heme-binding globin-NOSoxy-NOSred arrangement, exhibiting profound similarity with the mammalian NOS (Correa-Aragunde et al. 2018). This globin domain likely plays a role in enhancing NO homeostasis and optimizing nitrogen utilization in cyanobacteria, as observed in various photosynthetic organisms (Del Castello et al. 2020). This cluster comprises both unicellular, filamentous, and heterocytous strains belonging to Synechococcales, Oscillatoriales, and Nostocales, respectively. Cluster V consists of FRQ1 and EF-hand calcium-binding motif on N-terminal of NOSoxy domain represented by the members of Microcoleus. Surprisingly, cluster VI contains the globin-NOSoxy-NOSred domains and an additional LCIB_C_CA (limiting CO2-inducible proteins B/C beta carbonic anhydrase) domain possibly involved in CO2 concentrating mechanisms (Fig. 3). Carbonic anhydrases (CA) convert CO2 and H2O to bicarbonate and H+. Recent findings suggest that CA can also bind NO2, leading to NO generation under acidic pH conditions in animal cells. This links energy metabolic products CO2/H+ to the production of vasoactive NO (Aamand et al. 2009; Capasso and Supuran 2024; Maia and Moura 2014). While the precise role of this domain in cyanobacteria remains unclear, it presents a captivating avenue for exploring unique catalytic properties within cyanobacterial NOS. Moreover, the absence of these domains in animal NOS, like the NAD(P)H-nitrite reductase, CO2-inducible proteins B/C beta carbonic anhydrase, and globin domain, is a tailored adaptation. These domains, pivotal for nitrogen and carbon metabolism in photosynthetic organisms, take a backseat in animals. Instead, animal NOS relies on domains like Ferredoxin-NADP reductase and calmodulin for redox maintenance and cellular regulation, contributing to a unique metabolic symphony. This strategic selection of domains not only highlights the nuanced evolutionary adaptations in NOS proteins but also emphasizes the efficiency of evolution in fine-tuning these proteins for optimal functionality across diverse biological landscapes. Additionally, the phylogenetic reconstruction revealed the intermixing of NOS genes across diverse taxonomic orders. The prevalence of NOS within Nostocales and Oscillatoriales members indicated that the presence of this gene is favored by cyanobacteria with larger genomes and complex morphologies. Furthermore, the absence of this gene in relatively simpler cyanobacterial strains encompassed within Gloeobacteriales, Pleurocapsales, Spirulinales, and Chroococcidiopsidales supports this assumption. In sum, these observations highlight the evolutionary dynamics of cyanobacterial NOS, showcasing adaptability to diverse environments through varied protein architectures.

Fig. 3
figure 3

Phylogenetic tree of putative cyanobacterial NOS proteins. NOS sequences from different cyanobacteria were extracted from the NCBI database and aligned with MUSCLE software (Edgar 2004). The evolutionary relationship was inferred using the Maximum likelihood method based on the JTT matrix-based model. The tree is drawn to scale, with branch lengths in the number of substitutions per site to infer the phylogenetic tree. This analysis involved 68 amino acid sequences. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Sequences indicated by an asterisk correspond to NOS from Scytonema sp. UIC 10036. The main cluster divisions are indicated by different colors. The different domain rearrangements obtained for each cluster are shown with Illustrator of Biological Sequence 1.0. CaM: calmodulin-binding site; EFh: EF-hand, Ca+2-binding motif; Fer2: BFD-like [2Fe-2S]-binding domain; LCIB_C_CA: limiting CO2-inducible proteins B/C beta carbonic anhydrase

Full size image

Dynamics of Calcium and Cofactors in Cyanobacterial Nitric Oxide Synthases

The calmodulin (CaM)-binding sequence observed in animal and algal NOS assumes a pivotal role, serving as a crucial link between the NOSoxy and NOSred domains. This connection regulates the reduction of NOSoxy by NOSred in response to Ca+2 (Aoyagi et al. 2003; Picciano and Crane 2019). By relying on CaM-mediated regulation, cells can finely tune NO production in response to intracellular Ca+2 dynamics. This precision is crucial for adapting NOS activity to various physiological conditions, contributing to the versatility of NO signaling in diverse cellular processes. CaM, composed of four EF-hands organized in two pairs, binds a Ca+2 ion in each hand (Barret et al. 2023). An interesting observation is the presence of the EF-hand motif in many cyanobacterial NOSs, particularly those in clusters II, III, and V (Fig. 3). This suggests the retention Ca+2 requirement for NOS activity in cyanobacteria, a characteristic that may have been lost in many prokaryotic NOS (Sudhamsu and Crane 2009). However, the complexity arises as despite the presence of this motif certain NOS isoforms exhibit Ca+2 independence, challenging our understanding. In contrast, the NOS from Synechococcus sp. PCC 7335, despite lacking a conventional Ca+2-binding site, still demonstrates a reliance on Ca+2 ions for its activity (Picciano and Crane 2019). This apparent contradiction prompts a deeper assessment of the nuanced interplay between the structural elements and functional dynamics within cyanobacterial NOS. Moreover, an essential question arises about the potential modulation of EF-hand-containing NOS activity in cyanobacteria by varying Ca+2 levels. The significance of Ca+2 in cyanobacterial physiology extends to multifaceted processes, including signal-mediated heterocyst differentiation under nitrogen-deficient conditions (Walter et al. 2016, 2020), adaptation to nitrogen starvation (Leganes et al. 2009), and regulation of growth and polysaccharide synthesis (Shi et al. 2013). Therefore, investigating NOS activity modulation by Ca+2 could reveal novel regulatory mechanisms and expand our understanding of NOS roles in cyanobacterial physiology.

Furthermore, both mammalian and bacterial NOS rely on distinct pterin cofactors: tetrahydrobiopterin (BH4) for mammalian NOS and tetrahydrofolate (THF) for bacterial NOS (Crane et al. 2010; Stuehr 1999). However, SyNOS (the NOS enzyme in cyanobacteria) exhibits a preference for BH4 over THF as a cofactor. THF is a molecule that contains a bulkier R side chain (glutamyl p-amino benzoic acid, pABA) than BH4 and tetrahydromonapterin (MH4), needing more physical space in the NOS pterin pocket. A recent study has also indicated the potential role of MH4 as a cofactor in SyNOS due to its structural similarity to BH4 (Correa-Aragunde et al. 2022). Cyanobacteria, recognized for their elevated levels of pterin glycosides, exhibit notable structural variations in both pterin and glycosyl moieties (Lee et al. 1999; Hanaya and Yamamoto 2013). These microorganisms possess the enzymatic machinery required for the biosynthesis of crucial cofactors, including BH4 and THF. The enzymatic cascade responsible for BH4 synthesis involves GTP cyclohydrolase I (GCHI), which catalyzes the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate—a precursor in the BH4 biosynthetic pathway (Kim and Park 2010). In THF biosynthesis, cyanobacteria utilize dihydrofolate reductase (DHFR) to reduce dihydrofolate to tetrahydrofolate, the biologically active form of folate. Furthermore, in BH4 biosynthesis, the enzyme sepiapterin reductase plays a pivotal role in converting MH4 to BH4 (Wang et al. 2016; Wakabayashi et al. 2020). Moreover, cyanopterin can be synthesized from either MH4 or tetrahydroneopterin (NH4) precursors (Moon et al. 2010; Feirer and Fuqua 2017). Despite these elucidated enzymatic pathways for multiple pterins in cyanobacteria, their specific roles as potential cofactors for cyanobacterial NOS remain an intriguing and experimentally unexplored area, urging the need for thorough investigation and characterization. Utilizing bioinformatic tools and ligand docking analyses, we conducted an in-silico study to explore the potential cofactor requirements for NOS catalytic activity. The results of molecular docking revealed the capacity of NOSoxy variants from Nostoc linckia, Leptolyngbya, Synechococcus, and Aphanizomenon to interact with THF, MH4, BH4, and cyanopterin cofactors. The negative binding energy observed across all analyzed NOS variants indicated stable interactions (Fig. 4). The stability of the docked structures were likely facilitated through various interactions, including van der Waals interactions, alkyl interactions, and carbon–hydrogen bonding (Fig. 4). The observed versatility in cofactor binding may be attributed to the shared pterin ring among all cofactors, a crucial element for the electron donor process, despite differences in their alkyl (R) side chains (Crane et al. 2010; Werner et al. 2003). Importantly, the R side chain of different cofactors does not appear to influence the electron donor process during NOS activity, as indicated by different studies (Crane et al. 2010; Weisslocker-Schaetzel et al. 2017; Bird et al. 2002). The structural isomer of MH4, namely NH4, could potentially substitute BH4 in mammalian NOS catalysis under in-vitro conditions. Furthermore, MH4 has demonstrated its ability to replace BH4 as a cofactor in E. coli NOS activity. In essence, this in-silico exploration not only sheds light on the diverse cofactor interactions within cyanobacterial NOS but also lays the foundation for future experimental validations and deeper understanding of the NOS catalysis in these organisms. These findings open avenues for further research into the biochemical nuances of cofactor utilization, offering potential insights into the regulatory mechanisms governing NOS activity in cyanobacteria.

Fig. 4
figure 4

High-quality homology model was constructed in Swiss Model for NOSoxy domain for each protein using default parameters where human eNOS served as the template. The quality of constructed model was determined by Ramachandran plot using PROCHECK (Laskowski et al. 1993). The interaction of cofactors with the NOSoxy domain of different cyanobacteria was analyzed through in-silico molecular docking. The receptor molecule (NOSoxy) was prepared by adding polar hydrogen and Kollman charges. Further, the ligands (cofactors) were also prepared for docking in AutoDock tools 1.5.7. Finally, molecular docking was performed using AutoDock vina.exe. The dock complex was visualized in Biovia Discovery studio 2019 and UCSF chimera 1.13.1 (Petterson et al. 2004). A 3D Model of cofactor Tetrahydrofolate (THF), dock complex with Nostoc linckia NOS, and 2D map showing interacting residues. B 3D Model of cofactor Tetrahydromonapterin (MH4), dock complex with Leptolyngbya sp. NOS, and 2D map showing interacting residues. C 3D Model of cofactor Tetrahydrobiopterin (BH4), dock complex with Synechococcus PCC 7335 NOS, and 2D map showing interacting residues. D 3D Model of cofactor cyanopterin, dock complex Aphanizomenon flos-aquae 2012/KM1/D3 NOS, and 2D map showing interacting residues

Full size image

Evolutionary Tale of Plant NOS-Like Enzymes

In the mid-1990s, the first indications emerged suggesting that plants might possess a NOS-like enzyme akin to mammals, initiating two decades of intensive research (Culotta and Koshland 1992). Several arguments were presented in favor of this possibility, including the observation of NOS-like activity in plant extracts, the effectiveness of animal NOS inhibitors in reducing NO production in plants, the detection of immunoreactive proteins responding to mammalian NOS inhibitors, and the biological effects triggered by the expression of mammalian NOS in plant cells (Gas et al. 2009a, b). While initially proposed candidates for plant NOS-like enzymes faced skepticism, recent evidence has cast significant doubt on their existence (Chandok et al. 2003). A compelling argument against the presence of a NOS-like enzyme in plants was recently presented (Jeandroz et al. 2016). This investigation involved an extensive search for transcripts encoding NOS-like proteins using data from the 1000 plants (1KP) international consortium, which encompasses transcriptome sequencing data from more than 1000 plant species, spanning angiosperms, gymnosperms, ferns, mosses, and algae. The analysis entailed screening for sequences exhibiting similarities to the human neuronal NOS gene (nNOS) within the transcriptomes of 1087 land plants, as well as in publicly available land plant genomes. Notably, no typical mammalian NOS-like sequences were identified (Jeandroz et al. 2016). This implies that, if plants have NOS-like enzymes, they might differ significantly from the conventional mammalian NOS, both in terms of sequence and functional characteristics.

This investigation took a pivotal turn with the identification and characterization of NOS in Ostreococcus tauri, which exhibits structural and functional similarity to mammalian NOS. This finding challenges the prevailing assumption of NOS absence in the plant kingdom. A key point of interest is that OtNOS preserves all key domains present in mammalian NOS, which encompass the oxygenase and reductase domains, along with critical cofactor-binding sites such as heme, H4B, NADPH, FMN, and FAD. Another intriguing aspect of OtNOS is its ability to utilize both tetrahydrobiopterin (H4B) and tetrahydrofolate (THF) as cofactors, offering metabolic flexibility in NO production (Foresi et al. 2010). The biosynthesis of H4B in photosynthetic organisms remains unverified, raising questions about the sources of this cofactor in algae. Moreover, in contrast to the functions of mNOS, OtNOS appears to serve a distinct purpose in safeguarding the algae against high intensity light, particularly during the peak exponential growth phase. This indicates that OtNOS likely plays a vital role in counteracting oxidative stress induced by prolonged exposure to intense light, a critical adaptation for photosynthetic organisms. Furthermore, the expression of OtNOS in the model plant Arabidopsis thaliana has revealed its ability to enhance various physiological traits, including germination rates, growth under high-salinity conditions, and tolerance to drought and oxidative stress (Foresi et al. 2015). Overall, the discovery of OtNOS challenges preconceived notions about NOS enzymes being exclusive to animals and opens avenues for further exploration into the evolution, function, and regulation of NOS-like enzymes in higher photoautotrophs. In a recent study, 15 typical NOSs were identified in algal species, confirming earlier pioneering work on the functional NOS in the green alga Ostreococcus tauri (Jeandroz et al. 2016; Foresi et al. 2010, 2015). The distribution of these NOSs in green algae did not align with phylogenetic expectations, underscoring the unique evolutionary origins of algal NOS-like proteins compared to their animal counterparts, suggesting independent evolutionary pathways and distinct functions in algae compared to the roles of NOS enzymes in animals. Structural analyses of algal NOS-like proteins have also revealed interesting features specific to these proteins. Algal NOSs lack the N-terminal hook and the Zn/S cluster motif, pivotal for the homo-dimer interface in mammalian NOSs (Santolini et al. 2017). These structural differences further support the idea that algal NOS-like proteins may have evolved for specialized functions within algae (Weisslocker-Schaetzel et al. 2017). These proteins may not strictly conform to the conventional definition of NOSs but could exhibit unique biochemistry and functions.

The Subcellular Localization of NOS-derived NO in Photosynthetic Organisms

The evolution of eukaryotic cells is a significant milestone in the history of life on Earth, marked by the development of a more complex cellular architecture. This complexity is evident in the emergence of distinct biochemical microniches within eukaryotic cells, which have evolved into specialized cell organelles, including mitochondria, chloroplast, cell nucleus, and the endoplasmic membrane systems (Baum and Baum 2014). Many of these cell organelles can trace their origins to endosymbiotic associations with ancient prokaryotic cells. For instance, the thylakoid lumen and stroma within chloroplasts is considered as an evolutionary descendant of the thylakoid space and cytoplasm found in ancestral cyanobacteria (Liebers et al. 2022). This endosymbiotic origin suggests the preservation of certain features from ancestral cyanobacterial biology in chloroplasts, including key aspects of ancient prokaryotic NO metabolism (Sato 2021). However, further development of subcellular NO biology in eukaryotes has necessitated adaptations and diversification. This includes the evolution of various NOS proteins, characterized by the incorporation of multiple domains, each conferring distinct properties and functions in higher life forms (Gupta and Mishra 2022).

The very first report identifying chloroplasts as a source of NO was discovered in tobacco leaf cells exposed to a fungal elicitor derived from Phytophthora cryptogea, along with a range of abiotic stressors (Foissner et al. 2000 et Confocal laser microscopy and electron paramagnetic resonance (EPR) showed that purified chloroplasts from soybean leaves actively participate in the synthesis of NO (Jasid et al. 2006). Chloroplasts incubated with 1 mM L-arginine showed a measurable NO production rate of 0.76 ± 0.04 nmol min−1 mg−1 protein. Co-incubation with animal NO synthase inhibitors ((L-NAME or L-NNA) led to a significant and noteworthy inhibition of NO production. Moreover, the reductive pathways involving nitrate/nitrite have been universally acknowledged as significant contributors to the regulation of NO levels in eukaryotic cells (Bender and Schwartz 2018). However, there are controversial data suggesting that chloroplast NO may exclusively originate from L-arginine (Tewari et al. 2013). Therefore, further investigations are essential to reconcile these conflicting findings and provide a comprehensive understanding of the origins of chloroplast NO. Moreover, herbicides like DCMU and paraquat significantly impacted the NO signal in chloroplasts (Galatro et al. 2013). As chloroplasts function as the central sites for carbon and nitrogen metabolism, along with the generation of ROS, the synthesis of NO within these organelles becomes pivotal in modulating diverse cellular signals. Excessive NO can disrupt chloroplast photosynthesis, impacting carbon fixation and triggering programmed cell death (Misra et al. 2014). Experiments involving exogenous NO application, using the donor molecule SNAP, demonstrated significant impacts on chloroplast functions. Specifically, NO inhibited photophosphorylation (Takahashi and Yamasaki 2002). Thus, maintaining tight control over NO levels within chloroplasts is imperative for proper cellular function.

Furthermore, another noteworthy subcellular location for NOS enzymes in photosynthetic organisms is within peroxisomes (Corpas et al. 2004). These membrane-bound organelles are recognized for their essential roles in various metabolic pathways, including lipid metabolism and detoxification. In plants, they contain antioxidant enzymes, including catalase, influencing NO-related processes, making them crucial for maintaining cellular redox balance (Sandalio and Romero-Puertas 2015) It is noteworthy that, at present, there are no documented reports regarding the presence of NR within plant peroxisomes, either enzymatic (i.e., nitrate reductase or xanthine oxidoreductase) or non-enzymatic. Nevertheless, an increasing body of experimental evidence collectively substantiates the existence of L-arginine-dependent NOS activity within these organelles (Barroso et al. 1999). This NOS activity adheres to the essential biochemical prerequisites, comprising NADPH, calmodulin, calcium, FAD, FMN, and BH4, aligning closely with the configuration observed in animal NOS (Stolz et al. 2002; Corpas et al. 2004; Loughran et al. 2005). Pharmacological and indirect genetic analyses provide additional lines of evidence supporting the presence of L-arginine-dependent NOS activity (Corpas et al. 2009). Additionally, there is evidence of the identification of peroxisomal proteins that undergo post-translational modifications (PTMs) stemming from NO, including both S-nitrosation and tyrosine nitration (Corpas and Barroso 2014). These plant peroxisomal proteins have been meticulously recognized as targets for NO-induced PTMs (reviewed in Corpas et al. 2021). Consequently, it is conceivable that NO, through PTMs such as S-nitrosation and/or nitration, serves as a pivotal component in orchestrating a sophisticated peroxisomal protein–protein network. This network could potentially encompass both synergistic and antagonistic interactions with other molecules. These molecular interactions are likely to play a central role in regulating peroxisomal functions and adapting to varying cellular conditions, underscoring the importance of exploring NOS-derived NO-mediated PTMs in peroxisomal biology.

Biological Functions

In the intricate tapestry of photosynthetic organisms, the role of NO emerges as a captivating symphony orchestrating a myriad of physiological processes. The versatility of NO synthesis, both enzymatically and non-enzymatically, unfolds a tale of its dual existence as an intracellular and extracellular messenger. As we delve into the realm of NO, its intricate involvement in developmental regulation, intercellular communication, stress surveillance, and programmed cell death within photosynthetic organisms becomes apparent. Recent discoveries spanning diverse organisms suggest that, although the generation of NO is a shared trait, the specific purposes behind NO production can exhibit significant variation. These three biological significances of NO, illuminated in the upcoming sections, reflect the key notes in this symphony, promising insights into the nuanced dynamics governing vital life processes within photosynthetic organisms.

How Does NOS-Generated NO Fit into Nitrogen Cycle Economy?

The escalating demand for agricultural food production has resulted in increased nitrogen fertilizer usage to attain higher yields. However, this excessive application has led to detrimental consequences, including nitrate leaching into groundwater causing eutrophication, soil degradation, and contributing to greenhouse gas emissions, exacerbating global warming and ozone depletion (Craswell 2021; Thalineau et al. 2016). In response to these challenges, enhancing Plant Nitrogen Use Efficiency (NUE) is imperative, defined as the ratio of plant seed yield to applied nitrogen. The prospect of harnessing NO metabolism/signaling for improved NUE has recently garnered attention. Researchers have strategically manipulated critical genes associated with various facets of nitrogen metabolism to enhance NUE in diverse plant species. NUE relies on both soil nitrogen availability and the plant’s effective utilization. The two essential components within plant NUE are Nitrogen Uptake Efficiency (NUpE) and Nitrogen Utilization Efficiency (NUtE), encompassing Nitrogen Assimilation Efficiency (NAE) and Nitrogen Remobilization Efficiency (NRE) (Rose and Wissuwa 2012; Santa-María et al. 2015).

NO in plants plays a dual role in nitrogen metabolism. Firstly, it functions as a signaling molecule, enhancing tolerance to nitrogen-deficiency stress by safeguarding photosynthetic pigments, and potentially serve as a nitrogen source. Secondly, NO facilitates the formation of symbiotic relationships between plant roots, rhizobia, and arbuscular mycorrhizal fungi (AMF), thereby improving plant access to nitrogen and phosphorus nutrients in the soil (Frungillo et al. 2014; Sun et al. 2015; Balotf et al. 2018; Shah et al. 2016 et Moreover, the application of NO has been documented to significantly enhance germination percentage, seedling growth, biomass accumulation, and yield across various vegetables, flowers, and fleshy fruits (Fan et al. 2013) Additionally, NO application demonstrates efficacy in delaying ripening, mitigating chilling injury, enhancing immunity, and increasing nutritional content. Remarkably, the gradual decline of NO levels during ripening, accompanied by protein nitration and nitrosation, underscores its role in these processes (González-Gordo et al. 2019). Moreover, in response to nutrient scarcity, NO functions as a growth regulator, stimulating root development and initiating ion translocation mechanisms to enhance mineral nutrient absorption (Buet et al. 2019).

Notably, L-arginine holds a critical role as an organic reservoir and nitrogen transporter in plants due to its high nitrogen-to-carbon (N/C) ratio. This makes nitrogen derived from L-arginine breakdown valuable for meeting the metabolic needs of various developmental stages, including vegetative growth and seed production (Ma et al. 2013; Chen et al. 2022). The ability of NOS to tap into the L-arginine nitrogen reservoir offers a promising avenue for redirecting these resources toward improved plant growth. Recent research by Del Castello and colleagues supports this notion, demonstrating significantly enhanced NUE and seed production in transgenic Arabidopsis plants expressing the NOS enzyme from cyanobacteria Synechococcus PCC 7335 (SyNOS) under both nitrogen-sufficient and nitrogen-deficient conditions (Del Castello et al. 2020). Remarkably, under nitrogen-limited conditions, elevated SyNOS expression is concomitant with upregulation of genes associated with nitrogen assimilation and an enhanced nitrogen remobilization process crucial for seed development. Moreover, SyNOS-expressing lines exhibit increased tolerance during nitrogen deficiency as compared to control plants (Del Castello et al. 2021). Similarly, heterologous expression of OtNOS in tobacco results in an enhancement of growth rate, flower quantity, and seed yield, but exclusively under nitrogen-sufficient conditions. Notably, the observed outcomes in OtNOS lines may result from factors beyond elevated NO levels, such as L-arginine depletion and potential increment in L-citrulline (Nejamkin et al. 2020). Moreover, the regulation of NO homeostasis, encompassing oxidation processes and enzymatic activities, including phytoglobin function, protein interactions, and cellular redox status, may contribute to these effects (Umbreen et al. 2018). Crucially, OtNOS represents a canonical NOS enzyme that lacks the globin domain, distinguishing it from SyNOS, which possesses a globin domain and exhibits the ability to oxidize NO to NO3. While OtNOS necessitates nitrogen sufficiency for its effects, it still holds promise for benefiting plants under specific conditions (Weisslocker-Schaetze et al. 2017). Contrastingly, SyNOS excels in nitrogen-deficient environments by enhancing nitrogen utilization efficiency and bolstering tolerance to such conditions (Correa-Aragunde et al. 2018; Picciano and Crane 2019). These distinctions underscore the potential applications of both NOS in optimizing plant growth and nutrient utilization in distinct contexts. Furthermore, proteins considered essential in the regulation of NUE undergo post-translational modifications mediated by NO. These modifications have been observed in key proteins like glutamine synthetase (Silva et al. 2019), as well as S-nitrosoglutathione Reductase 1 (GSNOR1) (Frungillo et al. 2014; Guerra et al. 2016). Intriguingly, NO also has the capacity to influence the expression of specific genes linked to NUE (Buet et al. 2022; Balotf et al. 2018). While there is a growing body of evidence supporting the regulatory role of NOS in nutrient-related processes, the importance of enriching nitrogen metabolism through unconventional enzymes, such as a unique NOS found in cyanobacteria, is still not well-explored. To advance in this field, it is important to identify the NOS proteins and their specific NO targets operating within the signaling network. This knowledge is essential for developing strategies aimed at enhancing NUE while minimizing unintended side effects (abiotic stresses, like salinity and cold) (Gull et al. 2019). Figure 4 presents a schematic model elucidating the intricate dynamics of NO signaling and metabolism aimed at improving NUE. The introgression of NOS may serve as a potential strategy to ameliorate these adverse effects, thereby fostering growth and facilitating the accumulation of essential nutrients, and enhancing the uptake of N, P, and/or K in specific plants, under particular growth conditions (Sun et al. 2015). Furthermore, the identification of transcription factors and structural proteins that come into play downstream within the NO signaling network, especially under specific nutrient stress conditions, opens up opportunities for a diverse range of methodologies (Fig. 4). These could involve the expression or editing of proteins that contribute to traits enhancing both Nitrogen Assimilation Efficiency (NAE) and Nitrogen Utilization Efficiency (NUtE). (Fig. 5)

Fig. 5
figure 5

NOS-derived NO-mediated strategies for enhancing Nitrogen Use Efficiency in challenging agricultural environments. NO signaling and metabolism improve NUE amid the environmental repercussions of excessive fertilizer use, increased nitrate (NO3.−) infiltration, and runoff/leaching. Transgenic plants expressing Nitric Oxide Synthase (NOS), like SyNOS, exhibit enhanced NUE and increased tolerance to nitrogen deficiency, suggesting a strategic approach for improving crop productivity under nitrogen-related challenges. Additionally, when confronted with abiotic stresses, NO emerges as a promising tool to mitigate adverse effects on growth, photosynthetic efficiency, and nitrogen translocation. NO also influences the expression of transcripts related to NUE. Future research directions involve identifying key factors in the NO signaling network to enhance Nitrogen Assimilation Efficiency (NAE) and Nitrogen Utilization Efficiency (NUtE)

Full size image

NOS-Generated NO: A Driving Force for Stress Alleviation

NO was first observed in photosynthetic organisms by Klepper through herbicide treatments, earlier than in animals. Since then, a series of studies have significantly deepened our understanding of NO as a non-traditional regulator, exerting a profound influence on various aspects of growth and development in photoautotrophs (Lamattina et al. 2003; Wendehenne et al. 2004; Delledonne 2005; Besson-Bard et al. 2008; Tun et al. 2008; Wilson et al. 2008). Under abiotic stress conditions, plants generate NO, initiating a cascade of responses (Khan et al. 2023). Additionally, the application of exogenous NO enhances resilience to environmental stresses such as floods, heavy metals, and nutrient deficiencies, underscoring its role in stress adaptation (Brouquisse 2019). The stress resilience function of NOS in the photoautotrophs Ostreococcus tauri, Synechococcus PCC 7335, and Aphanizomenon flos-aquae 2012/KM1/D illustrates the critical role played by NOS-generated NO in enhancing the ability of these organisms to withstand and adapt to various environmental challenges.

NOS Activity in Stress Resilience: Case Studies of Ostreococcus tauri and Synechococcus PCC 7335

The precise roles of NO in the physiology of photoautotrophs have predominantly relied on two predominant approaches—exogenous application of NO donors/scavengers and exploration of nitrate/nitrite reductase pathways for NO generation. However, the discovery of a canonical NOS enzyme in the green algae Ostreococcus tauri has added a unique layer to our understanding of NO-related biology in photosynthetic organisms like cyanobacteria, algae, and plants. OtNOS activity exhibits variation with the growth phase, reaching an optimum level during exponential growth. This activity significantly increased in response to high-intensity, photo-inhibitory light conditions, suggesting a potential role for NO in mitigating oxidative damage induced by intense light, similar to observations in higher plants (Foresi et al. 2010). Furthermore, OtNOS expressed in Arabidopsis under the regulation of a stress-inducible promoter showed that the transgenic lines displayed several positive physiological effects. These included improved germination rates, enhanced root and shoot development under high-salinity conditions, and increased tolerance to drought. These findings underscore the potential significance of OtNOS-derived NO-related mechanisms in enhancing plant resilience and performance under adverse environmental conditions (Foresi et al. 2015). Likewise, introducing SyNOS into E. coli empowers the bacteria to flourish in minimal media where L-arginine serves as the sole nitrogen source. Moreover, this genetic modification elevates the growth rate of E. coli in comparison to the wild type, particularly under restricted nitrogen availability (Correa-Aragunde et al. 2018).

NOS Activity in Stress Resilience: Case Study of Aphanizomenon flos-aquae 2012/KM1/D3

Recently, a connection has been established between NOS activity and cyanobacterial physiology under stress responses in cyanobacterium Aphanizomenon flos-aquae 2012/KM1/D3 (Gupta and Mishra 2022). Specifically, the inhibition of NOS has been found to yield far-reaching consequences in the functioning of cyanobacteria. Notably, it leads to the suppression of photosynthetic efficacy, inducing significant shifts in carbon utilization patterns and alterations in fatty acid profiles. Additionally, NOS inhibition disrupts the typically favorable redox status within cyanobacterial cells, evidenced by reductions in the critical AsA/DHA and GSH/GSSG ratios, the major cyanobacterial redox couples. However, NOS elicitation leads to a substantial increase in intracellular NO levels within cyanobacterial cells. It is hypothesized that AfNOS, the NOS enzyme in Aphanizomenon flos-aquae, plays a pivotal role in counteracting the oxidative stress challenges by influencing the cyanobacterium’s antioxidant defense system. Consequently, this finely tuned regulatory mechanism ensures the maintenance of stable growth as the cyanobacterium Aphanizomenon transitions from the exponential to the stationary growth phase (Gupta and Mishra 2022; Gupta et al. 2024). Cumulatively, all these studies point toward the contention that NO, generated through the activity of NOS, functions as a molecular cue that triggers a cascade of defense mechanisms and, consequently, it orchestrates an NO-dependent response to a diverse array of stress conditions. Thus, examining the inherent protective mechanism of NOS-derived NO in photosynthetic organisms under environmental stresses represents a promising frontier for future research. The discovery of NOS genes in other photosynthetic organisms holds the potential to reveal previously uncharted roles for NO in their biological processes (Dato et al. 2015; Kumar et al. 2015; Jeandroz et al. 2016). The ramifications of this research will extend across a broad spectrum of applications, including agriculture, biotechnology, and environmental conservation.

NO Kidding: Maestro of Microbial Chatter in Photosynthetic Microorganisms

NO exhibits versatility in its synthesis, being enzymatically generated through NOS or NR, as well as non-enzymatically originating from nitrite, particularly within acidic compartments such as the apoplast of plant cells (Yamasaki et al. 1999; Jeandroz et al. 2016). Upon its production, NO possesses the ability to traverse cell membranes through simple diffusion, subsequently instigating a multitude of responses in neighboring cells (Bidle 2016; Astier et al. 2021). The capacity of NO to serve as both an intracellular and extracellular messenger underscores its central role in coordinating signaling pathways across a wide spectrum of species. It is intricately involved in processes ranging from developmental regulation to intercellular communication, stress surveillance, and the finely tuned regulation of programmed cell death (PCD) within photosynthetic organisms (Neill et al. 2003). The groundbreaking revelation of NO serving as a diffusible extracellular signal in aquatic settings gained prominence through its first proposal in diatoms, specifically Thalassiosira weissflogii and Phaeodactylum tricornutum. Within these diatoms, NO was recognized as a pivotal player in stress perception, potentially serving as a trigger for regulated cell death (RCD) in adjacent cells (Vardi et al. 2008; Bhattacharjee et al. 2021). Further, in the study conducted on cultures of the marine alga Emiliania huxleyi, intracellular production of NO was detected 24 h following viral infection (Schieler et al. 2019). This temporal observation precedes the onset of a ROS burst, a critical event established as an essential factor in PCD induction and subsequent host cell lysis. The study revealed the presence of extracellular nitric oxide (eNO) in the cell-free media post-infection, leading to the intriguing hypothesis that eNO functions as a signaling molecule, facilitating the communication of the infection event to neighboring cells (Bhattacharjee and Mishra 2020). This intriguing revelation led the authors to put forth a broader concept, emphasizing the exchange of inorganic nitrogen in the form of eNO between bacteria and photosynthetic microorganisms as a form of microbial communication that holds ecological significance, surpassing traditional kingdom boundaries (Abada et al. 2021). Similarly, the role of eNO extends to various stress responses in green algae, including Chlorella vulgaris, Chlamydomonas reinhardtii, and Scenedesmus obliquus (Mallick and Mohn 2000; Astier et al. 2020, 2021). Paradoxically, NO plays a pivotal role in promoting survival by serving as a protective agent against RCD in cyanobacteria. In Anabaena sp. PCC 7120, instances of iron deficiency result in the onset of oxidative stress, bringing forth inevitable consequences. NO emerges as a key player, efficiently mitigating pigment damage and promoting growth (Kaushik et al. 2016). Likewise, exposure to aluminum (Al) stress hinders growth and disrupts cellular processes. However, the eNO effectively reverses these adverse effects, resulting in increased cell viability (Chakraborty et al. 2019; Verma et al. 2018; Tiwari et al. 2019. In a separate study focusing on the cyanobacterium Nostoc muscorum ATCC 27893 subjected to nickel (Ni) stress, exogenous Ca2+ and NO supplementation efficiently mitigated Ni-induced damage by improving photosynthesis rate, nitrogen metabolism, and antioxidant defenses (Verma et al. 2021). Moreover, eNO efficiently alleviates damage induced by enhanced UV-B radiation in Spirulina platensis-794, ensuring the uninterrupted continuation of the photosynthetic process (Xue et al. 2007). Despite the valuable insights provided by these studies into NO’s multifaceted roles in physiological processes and stress responses among diverse photosynthetic microorganisms, the question of whether NO functions as a regulator of cell death or survival by diffusing through the aqueous solution remains unanswered. This unresolved aspect adds a layer of complexity to the multifaceted functionalities of NO, urging further exploration and detailed investigations into its precise mechanisms in diverse biological scenarios.

Advances and Challenges in NO Quantification

In spite of the extensive research on NO roles in metabolic pathways and signal transduction cascades, the precise quantification of NO levels remains challenging. Additionally, NO has an exceptionally short half-life; for example, at a 10 μM concentration, its half-life is around 80 s, but at 100 μM, it decreases to about 8 s (Wink and Mitchell 1998). This rapid mobilization from source to sink at higher concentrations, coupled with rapid autoxidation, compromises the accurate quantification of NO in biological systems. In the past, numerous review papers have undertaken thorough assessments of diverse NO measurement techniques, providing detailed insights into the strengths and limitations inherent in each approach (Csonka et al. 2015; Möller and Denicola 2019; Vishwakarma et al. 2019). The method for quantifying NO, limitations, and potential drawbacks are comprehensively outlined in Table 1. Here we will explore the challenges and intricacies of measuring NOS-derived NO within three distinct contexts: (1) complexity of subcellular NO detection, (2) scavengers and regulation of NO, and (3) limitation of SNO measurement. Examining the subcellular localization of NO is vital for comprehending its diverse biological functions. Nonetheless, this investigation is not without challenges, especially when employing diaminofluorescein (DAF) probes. These fluorescent probes are widely applied to measure NO formation within biological systems (Cortese-Krott et al. 2012). The mechanism of DAF probes revolves around the reaction between NO and the diaminofluorescein derivative, resulting in the formation of a highly fluorescent triazolofluorescein product. DAF probes such as DAF-2 and DAF-FM, initially non-fluorescent and cell-permeable, undergo a chemical transformation upon encountering NO, resulting in the generation of fluorescence for NO detection (Planchet and Kaiser 2006). What captivated numerous scientists in the NO field about these molecules is their apparent user-friendliness, especially when contrasted with other techniques that demand specialized instrumentation and expertise, such as chemiluminescence and electron paramagnetic resonance (Li et al. 2021). Notwithstanding their practicality, the reactivity and biological intricacies of cell-permeable DAF probes in the cellular milieu remain enigmatic. Post-reaction, these probes exhibit mobility within cells, potentially traversing different compartments, impeding precise localization. Variability in esterase activity and cell permeability adds complexity, yielding diverse probe distributions among cells. Factors such as reactive species or pH changes can influence local fluorescence, challenging accurate subcellular localization (Wang et al. 2000). Moreover, DAF probes may react with various molecules, including N2O3, or other nitrosating compounds, potentially yielding misleading signals. The presence of oxidants and ROS further complicates the formation of nitrosative species, potentially leading to DAF-2-T or DAF-FM-T formation even without increased NO, particularly in conditions like hypoxia and oxidative stress (Gupta and Igamberdiev 2013). Rigorous controls are imperative to discern nonspecific NO-independent signals. DAFs undergo secondary reactions with ascorbate. Moreover, alterations in temperature and light conditions can disrupt their reactivity with NO, posing challenges in accurately measuring NO levels (Li et al. 2021). Overcoming these limitations is paramount to achieving precise subcellular NO localization, thereby advancing our understanding of NO’s diverse functions within cells and its impact on various biological processes.

Table 1 Methodology for quantifying nitric oxide: sensitivity, procedure, advantages, and disadvantages
Full size table

Secondly, the half-life of NO is significantly modulated by the presence of scavengers, such as non-symbiotic hemoglobin (class 1 and class 2). This scavenging can vary between different species and growth conditions (Hill 2012). In Arabidopsis plants, non-symbiotic hemoglobin proteins are found to be responsible for turning atmospheric NO into NO3 (Perazzolli et al. 2004). This process allows the plant to utilize atmospheric NO when nitrogen in the soil is scarce and helps clean the air by reducing harmful nitrogen oxides (NOx) (Kuruthukulangarakoola et al. 2017). Similarly, a recent study in cyanobacteria indicated that the globin domain of SyNOS acts in concert with flavohemoglobins to form NO3 (Correa-Aragunde et al. 2018). Flavohemoglobins are proteins found in bacteria that play a critical role in the conversion of NO into NO3 (Guo and Gao 2021). SyNOS encapsulates all the essential components within a single protein, creating a complex system capable of converting L-arginine into NO3 (Picciano and Crane 2019). The resulting NO3 can re-enter the cell’s nitrogen metabolism pathways and be converted back into amino acids by NR and NiR enzymes (Berger et al. 2020). Measuring NO directly provides valuable insights into SyNOS activity and NO production but relying solely on NO measurements may underestimate actual NO levels due to rapid scavenging by flavohemoglobins. Furthermore, the potential generation of NO3 by NR adds a layer of complexity to the interpretation of NOS-derived NO and NO3 levels. The coexistence of NO3 underscores the need for a clear differentiation between the two. Hence, a comprehensive understanding of the spatial and temporal aspects of NO and NO3 generation and accumulation is vital for advancing our knowledge of this complex system and its potential applications in agricultural practices. Concurrently, ongoing research focuses on novel cyanobacterial NOS homologs with a globin domain (cluster III), aiming to uncover global implications of NO in photosynthetic organisms. Precision in NO measurement remains a pivotal element for fostering a comprehensive understanding in this scientific pursuit.

Lastly, NO can also react with glutathione (GSH) to produce S-nitrosoglutathione (GSNO), which is considered a significant cellular reservoir of NO capable of forming protein-S-nitrosothiols (protein-SNO) (Frungillo et al. 2014). The detection of S-nitrosothiols is challenging and prone to many artifacts (Diers et al. 2014). The Saville–Griess assay provides a quantitative assessment of total SNO levels but lacks the ability to distinguish specific SNO species. In cysteine-blocked spectrophotometry, complete blocking of free cysteine thiols is essential for accurate measurements, and incomplete blocking may result in inaccurate SNO quantification (Rőszer and Rőszer 2012). EPR spectroscopy and Mass spectrometry methods, such as LC–MS/MS, require sophisticated equipment and expertise, making them less accessible in some laboratories (Hao et al. 2006). The chemiluminescence resulting from the ozone-mediated oxidation of SNO initially appeared promising as it met the required criteria. However, the presence of a multitude of different NO metabolites in biological milieu can produce chemiluminescent signals, which may lead to false positives or require additional specificity controls (Nagababu et al. 2006). The biotin-switch technique, employed for the detection of RSNO, has significant limitations, including incomplete thiol-blocking before reduction/labeling and the specificity of reducing agents for RSNO (Diers et al. 2014 Recently, a versatile PBZyn SNO probe has been developed for characterizing SNO; however, its applicability in diverse biological samples remains to be explored (Clements et al. 2020). Commercial kits for detecting protein S-nitrosation are also available but it is crucial to note that merely identifying S-nitrosated proteins is inadequate to assign a biological role, as some modifications may be functionally inert or the extent of thiol modification is insufficient to impact that particular metabolic or signaling pathway. The ongoing efforts to develop and refine detection methods and enhance protocols for mitigating artifacts are key steps toward improving the precision and reliability of NO quantification in various organisms, including cyanobacteria. Therefore, it is recommended to identify the optimal combination of methods tailored to the specific objectives of the study at hand.

Conclusion and Future Issues

The evolution of NOS unfolds as a dynamic saga intertwined with Earth’s ancient history, showcasing adaptability and convergent evolution. The comprehensive analysis of cyanobacterial NOS reveals rich structural diversity across distinct clusters, shaped by environmental cues. Cyanobacterial NOSs consisting of EF-hand motifs for calcium dynamics and versatile pterin cofactor interactions provided valuable insights into the versatile functionality. The discovery of functional NOS-like enzymes in plants, as seen with OtNOS, challenges the initial skepticism. Additionally, unique structural features of algal NOSs emphasize their distinct pathways in photosynthetic organisms. Subcellular localization emphasizes pivotal roles within chloroplasts and peroxisomes, reflecting the complexity of NO metabolism and its impact on cellular implications. NOS-derived NO exhibits multifaceted functions, influencing plant growth and development, stress responses, microbial communication, and cell fate regulation, offering a comprehensive understanding for diverse applications in agriculture, biotechnology, and environmental management.

The iterative bioinformatics and experimental studies on NOS raise several questions such as why are NOSs limited to specific subsets of cyanobacteria? Why are they present in certain species within a genus and not in others? Additionally, what is the evolutionary connection between animal and prokaryotic NOS? Moreover, the utilization of pterin glycosides, with diverse roles in cyanobacteria, as potential cofactors for NOS activity remains unexplored. Furthermore, how do cofactor requirements and redox partners contribute to their function and regulation? Although some variation in electron sources and cofactor relays among prokaryotic NOSs is anticipated, the degree of this variation and its impact on biological activity remain uncertain. Could NOS isoforms undergo differential regulation under various stress conditions? Moreover, some cyanobacterial NOSs are found with dedicated reductases. This challenges the prevailing hypothesis of the existence of a NOSoxy domain followed by the emergence of the NOSred domain. The appearance of cyanobacterial NOS breaks this paradigm and opens new directions regarding NOS evolution in eukaryotes. As ongoing investigations deepen our insights, we anticipate future contributions from researchers, propelling the field into uncharted realms of understanding. The dynamic nature of this evolving field promises continual exploration, pushing the boundaries of our knowledge and opening avenues for innovative discoveries in NOS-derived NO biochemistry.