Nitrogen fixation, the conversion of atmospheric nitrogen (N2) into ammonia (NH3) or related nitrogenous compounds, is a vital process for sustaining life on Earth, particularly in the context of plant growth and agricultural productivity (Smil, 2002). While the majority of nitrogen fixation occurs through microbial processes, certain plants, known as nitrogen-fixing plants, have evolved symbiotic relationships with nitrogen-fixing bacteria to facilitate this crucial biochemical reaction (Vance et al., 2001). The phenomenon of nitrogen fixation in plants has fascinated researchers for decades, driving extensive investigations into the molecular mechanisms underlying this complex biological process. Understanding these mechanisms is of utmost importance not only for elucidating fundamental aspects of plant biology but also for enhancing agricultural sustainability through the development of nitrogen-fixing crops. At the heart of nitrogen fixation in plants lies the symbiotic association between the plant host and nitrogen-fixing bacteria, typically belonging to the genera Rhizobium, Bradyrhizobium, or Frankia. This symbiosis is established through intricate molecular signaling pathways that enable the recognition, colonization, and accommodation of the bacterial partner within specialized plant structures called nodules (Gibson et al., 2008). Key to the establishment of nitrogen-fixing symbiosis is the exchange of signaling molecules between the plant and bacterial partners. Among these molecules, the plant-derived flavonoids play a crucial role in initiating the nodulation process by inducing the expression of nodulation (nod) genes in compatible rhizobia (Kosslak et al., 1984). In response, rhizobia produce specific nodulation factors (Nod factors) that trigger a series of molecular events leading to nodule formation (Dénarié  et al., 1996). Once nodules are formed, nitrogen fixation occurs within specialized structures called bacteroids, which are bacterial cells that have undergone differentiation in response to plant signals. Within the bacteroids, the enzyme nitrogenase catalyzes the conversion of atmospheric nitrogen to ammonia, a process that requires high energy expenditure and is tightly regulated to prevent the wasteful consumption of resources. In addition to symbiotic nitrogenfixation, certain non-leguminous plants, such as cereals and grasses, are capable of hosting endophytic nitrogen-fixing bacteria within their tissues. This alternative mode of nitrogen fixation presents another avenue for exploring the molecular mechanisms underlying this process and holds promise for improving nitrogen utilization efficiency in agricultural systems (Reinhold-Hurek et al., 2015). Advances in molecular biology, genomics, and bioinformatics have greatly accelerated our understanding of the genetic basis of nitrogen fixation in plants (Gresshoff et al., 2015). Genome sequencing efforts, coupled with functional genomics approaches, have led to the identification and characterization of numerous genes involved in nodulation, nitrogen transport, and nitrogen assimilation pathways. Despite significant progress, many aspects of nitrogen fixation in plants remain poorly understood, presenting exciting opportunities for further research (Ferguson et al., 2010). Elucidating the intricate signaling networks, regulatory mechanisms, and metabolic pathways involved in nitrogen fixation holds the key to unlocking the full potential of this process for sustainable agriculture and environmental stewardship (Galloway et al., 2008). In this review, we provide an overview of the current state of knowledge regarding the molecular mechanisms underlying nitrogen fixation in plants. We will discuss recent advances in our understanding of symbiotic and non-symbiotic nitrogen fixation, with a focus on the key genes, signaling pathways, and regulatory mechanisms involved. Additionally, we will explore the potential applications of this knowledge for improving nitrogen use efficiency and reducing the environmental impact of agricultural practices. By synthesizing findings from diverse disciplines, we aim to shed light on the multifaceted nature of nitrogen fixation in plants and inspire future research directions in this field. Nitrogen fixation, the conversion of atmospheric nitrogen (N2) into ammonia (NH3) or related compounds, is a fundamental biological process critical for sustaining life on Earth.  While traditionally associated with prokaryotes, such as diazotrophic bacteria and archaea, recent research has unveiled the presence of nitrogen-fixing capabilities in a select group of plants, particularly legumes, through symbiotic interactions with nitrogen-fixing bacteria known as rhizobia. This symbiotic relationship, established in specialized root organs called nodules, facilitates the exchange of nutrients between the plant host and the microbial partner. Moreover, emerging evidence suggests that non-leguminous plants, including cereals and other crops, may also possess the ability to perform nitrogen fixation independently or in association with endophytic microorganisms. Understanding the molecular mechanisms underlying nitrogen fixation in plants holds immense significance for agriculture and environmental sustainability. By reducing the reliance on synthetic nitrogen fertilizers, which are energy-intensive to produce and contribute to environmental degradation, harnessing plant-mediated nitrogen fixation has the potential to mitigate the negative impacts of conventional agricultural practices. However, unraveling the intricate molecular networks governing this process presents a formidable challenge due to its multifaceted nature and the involvement of diverse biological players. Key components of the nitrogen fixation pathway include nitrogenase enzymes responsible for catalyzing the reduction of atmospheric nitrogen, oxygen-scavenging mechanisms to protect nitrogenase from inactivation by oxygen, and signaling pathways orchestrating the establishment and maintenance of symbiotic associations. Recent advances in genomics, transcript omics, proteomics, and metabolomics have provided unprecedented insights into the molecular basis of nitrogen fixation in plants. Genome-wide studies have identified candidate genes involved in nitrogen fixation and elucidated their regulatory networks, shedding light on the genetic determinants of nitrogen-fixing efficiency and symbiotic specificity. Transcriptomic analyses have revealed dynamic changes in gene expression patterns during nodule development and function, highlighting the spatiotemporal regulation of nitrogen fixation-related genes. Proteomic and metabolomic studies have complemented these findings by characterizing the repertoire of proteins and metabolites associated with nitrogen fixation, offering valuable clues about the metabolic pathways and biochemical processes underlying nitrogen assimilation and transport. In addition to dissecting the molecular machinery of nitrogen fixation, researchers are also exploring strategies to enhance nitrogen-fixing capabilities in plants through genetic engineering and microbiome manipulation. Engineering nitrogen-fixing traits into non-leguminous crops holds immense potential for improving agricultural productivity and sustainability, thereby contributing to global food security. Furthermore, harnessing the beneficial interactions between plants and nitrogen-fixing microorganisms offers promising avenues for developing novel biofertilizers and sustainable agricultural practices.

BIOLOGICAL NITROGEN FIXATION

Biological nitrogen fixation is a crucial process in the nitrogen cycle, wherein atmospheric nitrogen (N2) is converted into ammonia (NH3) by nitrogen-fixing microorganisms such as bacteria and archaea (Poole et al., 2018). This process plays a fundamental role in maintaining soil fertility and sustaining plant growth in various ecosystems (Gaby & Buckley 2017). Overview of biological nitrogen fixation reveals its significance in agriculture, as it reduces the reliance on synthetic nitrogen fertilizers, which can lead to environmental pollution and contribute to greenhouse gas emissions. Nitrogen fixation occurs through various mechanisms, including free-living bacteria such as Azotobacter and Clostridium, symbiotic associations with leguminous plants, and mutualistic interactions with diazotrophic bacteria in non-leguminous plants like rice and sugarcane (Mus et al., 2016). Nitrogen fixation symbiosis involves a mutualistic relationship between nitrogen-fixing bacteria, primarily belonging to the genera Rhizobium, Bradyrhizobium, and Azospirillum, and their plant hosts, where the bacteria colonize the plant roots and form specialized structures called nodules. Within these nodules, nitrogenase enzyme complexes catalyze the reduction of atmospheric nitrogen to ammonia, providing the plant with a vital source of nitrogen for growth and development. The nitrogenase enzyme complex comprises two component proteins: the nitrogenase reductase (Fe protein) and the nitrogenase iron protein (MoFe protein), which work together to convert nitrogen gas into ammonia through a complex series of reactions that require ATP and a reducing agent (Ribbe & Burgess 2001; Einsle et al., 2002). In summary, biological nitrogen fixation is a crucial process mediated by diverse microorganisms, with significant implications for agriculture, ecosystem sustainability, and environmental management (Falkowski et al., 2008). Nitrogen fixation symbiosis, a pivotal process in the nitrogen cycle, involves a mutualistic relationship between nitrogen-fixing bacteria and host plants, facilitating the conversion of atmospheric nitrogen into biologically useful forms. This intricate symbiosis is extensively studied across various disciplines, including microbiology, plant biology, and ecology (Smith et al., 2019). One of the key players in this symbiotic interaction is the nitrogenase enzyme complex, responsible for catalyzing the conversion of atmospheric nitrogen (N2) to ammonia (NH3), which can be assimilated by plants. The nitrogenase enzyme complex comprises multiple protein components, including the MoFe protein (dinitrogenase) and the Fe protein (dinitrogenase reductase), which work in concert to carry out the nitrogen fixation process (Hoffman et al., 2014; Howard et al., 2006). These proteins are encoded by nif genes, which are often organized into operons and regulated in response to environmental cues, such as nitrogen availability and oxygen levels. The assembly and activity of the nitrogenase complex are tightly regulated to ensure efficient nitrogen fixation while minimizing energy expenditure and potential damage by reactive oxygen species (Robson et al., 1980). In nitrogen-fixing symbioses, such as those formed between leguminous plants and rhizobia, the nitrogenase enzyme complex operates within specialized structures called nodules, which provide a low-oxygen environment conducive to nitrogen fixation. The establishment and maintenance of these symbiotic associations require complex signaling events between the host plant and the symbiotic bacteria, involving the exchange of molecular signals, such as flavonoids and nodulation factors (Ferguson et al., 2010; Perret et al., 2000). Furthermore, the efficiency of nitrogen fixation symbiosis is influenced by various factors, including the genetic background of both the host plant and the symbiotic bacteria, as well as environmental conditions such as soil pH, temperature, and moisture (Mus et al., 2016). Understanding the molecular mechanisms underlying nitrogen fixation symbiosis and the functioning of the nitrogenase enzyme complex is crucial for improving agricultural sustainability and reducing dependence on synthetic nitrogen fertilizers. In conclusion, nitrogen fixation symbiosis involving the nitrogenase enzyme complex is a fascinating biological phenomenon with significant implications for both natural ecosystems and agricultural systems. Through interdisciplinary research efforts, scientists continue to unravel the complexities of this symbiotic interaction, paving the way for novel strategies to enhance nitrogen fixation efficiency and mitigate environmental impacts associated with nitrogen fertilizer use (Galloway et al., 2008).

Fig. 1. Schematic representation of biological nitrogen fixation (BNF). A For effective symbiotic association development mainly two classes of genes are required: nodulation and nitrogen fixation genes. Nodulation genes, e.g. nod ABC, interact with host-specific nodulin genes and plant flavonoids. Nitrogen fixation genes possess the structural genes (nif and fix) to encode nitrogenase (nifHDK) enzyme that is the lead actor for nitrogen fixation; b metabolic reaction of BNF is highly expensive as it consumes 16 ATP to fix one molecule of N; c the key actor nitrogenase genes assembly-the genes are coloured by their function as blue (nitrogenase), green (cofactor biosynthesis, shading corresponds to operons), and yellow (e⁻ transport) and a horizontal bar indicates overlapping genes; d mature nitrogenase enzyme with electron-transport chain catalyses nitrogen fixation process.

Molecular Players in Nitrogen Fixation. Nitrogen fixation, the biological conversion of atmospheric nitrogen into ammonia, is a vital process for sustaining life on Earth (Bergman et al., 2007). At the heart of this process lies a sophisticated interplay of molecular players orchestrated by diverse organisms, including bacteria and archaea. The nitrogenase complex, a multi-subunit enzyme, catalyzes the reduction of atmospheric nitrogen to ammonia. Comprised of the MoFe protein and the Fe protein, this intricate machinery is central to nitrogen fixation. The Fe protein mediates the transfer of electrons from ferredoxin to the MoFe protein, where nitrogen reduction occurs. Iron-sulfur (Fe-S) clusters serve as essential cofactors in nitrogenase, facilitating electron transfer reactions. These clusters play crucial roles in electron transport and substrate binding within the nitrogenase complex (Lancaster et al., 2020). Their unique redox properties enable the conversion of molecular nitrogen to ammonia (Kim et al., 2015).The molybdenum cofactor (Moco) is indispensable for the catalytic activity of nitrogenase (Seefeldt et al., 2020). Moco acts as the site for nitrogen binding and reduction, playing a pivotal role in the enzymatic mechanism.  Its intricate structure and coordination chemistry are critical for nitrogen fixation Numerous regulatory mechanisms govern nitrogen fixation to optimize energy expenditure and adapt to varying environmental conditions. Transcriptional regulators, such as NifA and NifL, modulate nitrogenase expression in response to cellular nitrogen levels (Ninfa & Jiang 2005). Post-translational modifications, including reversible ADP-ribosylation, further fine-tune nitrogenase activity (Moure et al., 2021). In symbiotic nitrogen fixation, leguminous plants form associations with nitrogen-fixing bacteria, such as Rhizobia. Signaling molecules, such as flavonoids and Nod factors, facilitate host recognition and nodulation (Madsen et al., 2003). The establishment of symbiotic relationships relies on a complex molecular dialogue between plant and bacterial partners (Roy et al., 2013). Nitrogen fixation significantly impacts global nitrogen cycling and ecosystem functioning (Vitousek et al., 2013). Anthropogenic activities, such as fertilizer use and industrial processes, have altered nitrogen fixation rates, leading to environmental consequences (Galloway et al., 2008). Understanding the molecular basis of nitrogen fixation is crucial for mitigating nitrogen-related environmental issues. In conclusion, nitrogen fixation is a complex biological process driven by an array of molecular players  from the nitrogenase complex to regulatory networks and symbiotic interactions, understanding these molecular mechanisms is essential for addressing global nitrogen challenges. Further research into the molecular intricacies of nitrogen fixation holds promise for sustainable agriculture and environmental conservation.

Nitrogenase Gene Regulation. Nitrogenase gene regulation is a highly intricate process governed by a myriad of factors, including environmental cues and cellular signaling pathways. In diazotrophic bacteria, such as Azotobactervinelandii and Klebsiellapneumoniae, nitrogenase gene expression is tightly regulated at multiple levels to ensure optimal nitrogen fixation while minimizing energy expenditure. At the transcriptional level, nitrogenase gene expression is controlled by a complex interplay of activators, repressors, and global regulators, including NifA, NifL, and NtrC, which respond to intracellular nitrogen and oxygen levels (Martinez-Argudo et al., 2020). Furthermore, post-transcriptional modifications mediated by small RNAs and RNA-binding proteins modulate nitrogenase gene expression in response to changing environmental conditions (Martinez-Argudo et al., 2020). Nitrogenase activity is also subject to allosteric regulation by metabolic intermediates, such as ATP, ADP, and Mg2+ ions, which fine-tune the enzyme's catalytic efficiency in response to cellular energy status. Additionally, post-translational modifications, such as phosphorylation and ADP-ribosylation, play crucial roles in modulating nitrogenase activity in various bacterial species (Martinez-Argudo and Little 2020). Environmental factors, including nitrogen availability, oxygen tension, and carbon/nitrogen ratios, exert profound effects on nitrogenase gene expression and activity through intricate signaling networks involving two-component systems, alternative sigma factors, and quorum sensing pathways (Martinez-Argudo et al., 2020). Furthermore, symbiotic associations between nitrogen-fixing bacteria and host plants involve sophisticated regulatory mechanisms that coordinate nitrogenase gene expression with plant developmental cues and nutrient demands (Martinez-Argudo et al., 2020). Overall, nitrogenase gene regulation represents a paradigm of cellular adaptation to environmental fluctuations, integrating transcriptional, post-transcriptional, translational, and post-translational regulatory mechanisms to optimize nitrogen fixation while minimizing energy expenditure.

Nodulation Genes and Signaling Pathways. Nodulation genes play a pivotal role in the establishment of nitrogen-fixing symbiosis between leguminous plants and nitrogen-fixing bacteria, such as Rhizobia. These genes encode proteins involved in various stages of nodule development, including recognition, colonization, and maintenance of the symbiotic relationship (Madsen et al., 2003). The nodulation process is initiated by the perception of rhizobial signaling molecules, called nod factors, by the plant's LysM receptor-like kinases (Limpens et al., 2003; Radutoiu et al., 2007). This perception triggers a signaling cascade leading to calcium spiking and activation of downstream transcription factors, such as Nodulation Signaling Pathway 1 (NSP1) and NSP2 (Kaló et al., 2005).Subsequently, NSP1 and NSP2 form a complex with another transcription factor, Nodule Inception (NIN), which activates the expression of early nodulation genes, including Nodulation Factor Receptor 1 (NFR1) and NFR5 (Schauser et al., 1999; Radutoiu et al., 2007). These receptors are essential for the perception of nod factors and the initiation of downstream signaling events leading to nodule organogenesis. Additionally, several other genes, such as Nodule Autoregulation Receptor Kinase (NARK) and CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides, regulate nodule number and distribution by negative feedback mechanisms (Searle et al., 2003; Okamoto et al., 2009). Moreover, the symbiotic interaction between plants and rhizobia requires the activation of symbiosis-specific transcription factors, such as Nodule Pectate Lyase (NPL) and Ethylene Response Factor Required for Nodulation (ERN) (Penmetsa et al., 2008). These transcription factors regulate the expression of genes involved in nodule development, including those encoding enzymes for nitrogen fixation, such as Nitrogenase (Nif) (Bauer et al., 1998). Additionally, several studies have highlighted the importance of microRNAs, such as miR169 and miR2111, in modulating nodulation by regulating the expression of nodulation-related genes. Furthermore, recent advances in molecular genetics and omics technologies have provided insights into the intricate regulatory networks governing nodulation. Transcriptomic analyses have revealed the dynamic changes in gene expression during different stages of nodulation, highlighting the roles of key regulators, such as NODULE INCEPTION (NIN) and CYCLOPS. Similarly, proteomic and metabolomic studies have identified key proteins and metabolites involved in symbiotic nitrogen fixation. Moreover, genetic studies in model legumes, such as Medicago truncatula and Lotus japonicus, have elucidated the functions of nodulation genes and signaling pathways. Overall, the intricate interplay of nodulation genes and signaling pathways orchestrates the establishment and maintenance of nitrogen-fixing symbiosis between leguminous plants and rhizobia. Understanding these regulatory mechanisms not only provides insights into plant-microbe interactions but also holds implications for sustainable agriculture and environmental sustainability.

Fig. 2. Genes and processes involved in early signaling during nodulation. (Iso) Flavonoids produced under low soil N (1) trigger the production of bacterial Nod Factors (2) that, together with other signals, are perceived by receptors at the plasma Membrane of epidermal cells (3). This triggers biochemical and physiological responses (4,5,6) that lead to changes in nuclear gene expression (7). See main text for key to Gene/protein names and further explanation of early signaling pathways and Components. M. truncatula protein names are provided unless otherwise specified.

PLANT-MICROBE INTERACTIONS IN NITROGEN FIXATION

Plant-microbe interactions play a crucial role in nitrogen fixation, a process essential for plant growth and ecosystem nitrogen cycling. One of the most well-studied symbiotic relationships involved in nitrogen fixation is the association between leguminous plants and nitrogen-fixing bacteria known as rhizobia, such as Rhizobium leguminosarum. Through the exchange of signals, including flavonoids from plants and nodulation factors from bacteria, the two partners establish a symbiotic relationship leading to the formation of root nodules where nitrogen fixation occurs. Within these nodules, the bacteria convert atmospheric nitrogen into ammonia, a form that plants can utilize for growth. Furthermore, other plant-associated microbes, such as mycorrhizal fungi, can also influence nitrogen fixation indirectly. Mycorrhizal fungi form symbiotic associations with plant roots, enhancing nutrient uptake, including nitrogen, and thus indirectly affecting nitrogen fixation rates. Additionally, non-symbiotic nitrogen-fixing bacteria, such as Azospirillum spp., can colonize the rhizosphere of various plants, promoting plant growth and potentially contributing to nitrogen fixation in agricultural systems (Bashan et al., 2004). The efficiency of nitrogen fixation in plant-microbe interactions can be influenced by various factors, including environmental conditions such as soil pH, temperature, and moisture (Martínez-Romero et al., 2010). Additionally, the genetic diversity of both the plant and microbial partners can impact the effectiveness of nitrogen fixation. Understanding the molecular mechanisms underlying these interactions is crucial for optimizing nitrogen fixation in agricultural systems and mitigating nitrogen pollution (Ferguson et al., 2010). Moreover, recent advances in molecular biology and omics technologies have provided insights into the intricate molecular dialogues between plants and nitrogen-fixing microbes (Larrainzar et al., 2015). Transcriptomic and proteomic studies have identified genes and proteins involved in the establishment and maintenance of symbiotic relationships, as well as the regulation of nitrogen fixation. Metagenomic approaches have also been instrumental In characterizing the diversity and functional potential of microbial communities associated with nitrogen-fixing plants. In conclusion, plant-microbe interactions in nitrogen fixation are complex and dynamic processes that involve a multitude of microbial and environmental factors. Understanding these interactions at the molecular level is crucial for sustainable agriculture and ecosystem management. Moreover, continued research in this field will uncover new strategies for enhancing nitrogen fixation efficiency and reducing the environmental impact of nitrogen fertilizer use.

Fig. 3. Diagrammatic representation of plant growth-promoting bacteria (endophytic, rhizospheric, and epiphytic) and functional attributes in impacting plant growth, development, and abiotic stress.

Nitrogen fixation in plants, a process crucial for their growth and development, poses several challenges in understanding its intricate molecular mechanisms. The symbiotic relationship between plants and nitrogen-fixing bacteria, such as Rhizobia, involves complex signaling pathways and genetic interactions. For instance, the nodulation process, essential for nitrogen fixation in leguminous plants, requires a coordinated response between plant-derived signals and bacterial nodulation factors. Furthermore, the molecular dialogue between plants and nitrogen-fixing bacteria involves multiple layers of regulation, including the recognition of symbiotic partners and the activation of specific genes. Key players in this process include receptor-like kinases and transcription factors, which mediate signal transduction pathways leading to nodule formation and nitrogen fixation (Kelly et al., 2017). Understanding the intricate crosstalk between these molecular components is essential for unraveling the nitrogen fixation process in plants. Moreover, environmental factors, such as soil pH, nutrient availability, and stress conditions, can significantly influence nitrogen fixation efficiency. The adaptation of nitrogen-fixing symbioses to diverse ecological niches highlights the plasticity of these molecular mechanisms. Deciphering the molecular basis of such adaptations promises insights into enhancing nitrogen fixation in agricultural systems. Despite significant progress in elucidating the molecular mechanisms underlying nitrogen fixation, several challenges remain. For instance, the precise regulation of nitrogenase activity, a key enzyme complex involved in nitrogen fixation, is still not fully understood. Additionally, the genetic diversity of nitrogen-fixing symbionts presents challenges in engineering more efficient nitrogen-fixing associations (Sutton et al., 2020). Looking ahead, future perspectives in the field of nitrogen fixation research are promising (Ferguson et al., 2010). Advances in omics technologies, including genomics, transcriptomics, and metabolomics, offer unprecedented opportunities to unravel the molecular intricacies of nitrogen fixation (Jones et al., 2021). Integration of multi-omics data will enable comprehensive systems-level understanding of nitrogen fixation pathways in plants. Furthermore, synthetic biology approaches hold great potential for engineering nitrogen-fixing capabilities in non-leguminous crops. By harnessing knowledge of the molecular mechanisms underlying nitrogen fixation, researchers can develop novel strategies to improve nitrogen use efficiency in agriculture. Ultimately, unraveling the complexities of nitrogen fixation in plants will not only enhance our understanding of fundamental biological processes but also contribute to sustainable agriculture and global food security.