Stress-Responsive Regulatory Networks in Legume–Rhizobium Symbiosis: Implications for Climate-Smart Agriculture

Abstract

Legume–rhizobium symbiosis is fundamental to sustainable agriculture because it supplies biologically fixed nitrogen, improves soil fertility, and reduces reliance on synthetic fertilizers. However, abiotic and chemical stresses, including drought, salinity, flooding, temperature extremes, heavy metals, and organic pollutants, disrupt nodulation and biological nitrogen fixation, limiting crop productivity and ecosystem sustainability. This review synthesizes current knowledge of the regulatory networks that enable legume–rhizobium symbiosis to adapt to environmental stress. We discuss how stress influences symbiotic signaling, infection, oxygen homeostasis, nitrogenase protection, phytohormonal regulation, antioxidant defenses, exopolysaccharide production, and plasmid-mediated adaptation. We further highlight the roles of root nodule-associated microorganisms and microbial interactions in maintaining symbiotic stability under adverse conditions. Finally, recent advances in multi-omics, genome editing, synthetic biology, and microbial consortia are evaluated for their potential to improve stress-resilient bioinoculants. Collectively, this review emphasizes that resilience of the legume–rhizobium symbiosis is an integrated property of both plant and microbes and identifies key regulatory mechanisms that can be exploited to develop climate-resilient and sustainable agricultural systems.

1. Introduction

Environmental stresses are major constraints to legume productivity and biological nitrogen fixation (BNF), with profound implications for soil fertility, agricultural sustainability, ecosystem resilience, and global food security [1]. Legumes depend on root nodule symbiosis with rhizobia to fix atmospheric nitrogen, thereby reducing reliance on synthetic nitrogen fertilizers and improving soil fertility [2]. The main ecological cues that encourage the development of legume–rhizobium symbiosis are deficiencies in nitrogen and phosphorus, which activate nodulation signaling pathways and flavonoid production [3]. However, once symbiosis is established, rhizobial survival, infection, nodule development, and biological nitrogen fixation can be disrupted by additional environmental constraints, such as drought, salinity, flooding, temperature extremes, soil acidity, nutrient imbalance, and chemical contaminants [4]. Because they negatively impact the establishment, maintenance, or efficiency of the symbiosis rather than its ecological initiation, these anthropogenic and environmental factors are collectively referred to as stress conditions. However, abiotic stresses, including drought, salinity, heat, flooding, nutrient imbalance, acidity, and oxidative stress, together with chemical contaminants such as heavy metals, hydrocarbons, pesticides, and industrial pollutants, disrupt multiple stages of symbiosis, including rhizobial survival, root colonization, flavonoid-mediated Nod factor signaling, infection thread formation, bacteroid differentiation, and nitrogenase activity [5,6]. Numerous stages of symbiosis, such as rhizobial survival in soil, root colonization, NSP1/NSP2-regulated flavonoid biosynthesis and Nod factor signaling, infection production, bacteroid differentiation, and nitrogenase activity, are disrupted by these conditions [7]. These disruptions reduce nitrogen availability, impair crop productivity, and increase dependence on chemical fertilizers, contributing to soil acidification, eutrophication, greenhouse gas emissions, and the loss of beneficial soil microbial diversity [6,8].

Environmental stress also alters rhizosphere microbial communities and cellular redox homeostasis through excessive accumulation of reactive oxygen and nitrogen species (ROS/RNS), leading to membrane damage, impaired respiration, and reduced nitrogenase activity [9,10]. Climate change further intensifies these effects by increasing the frequency and intensity of drought, salinity, flooding, temperature extremes, and pollutant mobilization, while simultaneously disrupting plant carbon allocation, phytohormone signaling, and the microaerobic conditions required for efficient nitrogen fixation within nodules [11,12,13,14]. The resilience of the legume–rhizobium symbiosis under environmental stress depends on coordinated responses from both the host plant and rhizobia. The host’s genetic background, including its regulatory networks, developmental plasticity, and stress-responsive signaling pathways, together with the stress-adaptive mechanisms of rhizobia, determines nodulation efficiency and nitrogen fixation under adverse conditions [4,15]. Therefore, enhancing the resilience of legume–rhizobium symbiosis has become a key strategy for sustaining crop productivity and reducing agricultural dependence on synthetic nitrogen inputs under changing environmental conditions.

Although previous reviews have examined rhizobial stress tolerance, nodulation signaling, plant growth-promoting rhizobacteria, microbial inoculants, exopolysaccharides, antioxidant systems, and heavy metal tolerance individually [16,17], few have integrated these mechanisms into a unified framework describing root nodules as dynamic stress-responsive microenvironments. In particular, the coordinated regulation of symbiotic signaling, oxygen homeostasis, nitrogenase protection, phytohormonal crosstalk, antioxidant defense, exopolysaccharide production, plasmid-mediated adaptation, and interactions with root nodule-associated microorganisms under multiple environmental stresses remains insufficiently synthesized.

This review presents stress-resilient legume–rhizobium symbiosis as an integrated molecular and ecological adaptation system operating at the plant–microbe interface. Although nutrient deficiency, particularly nitrogen and phosphorus limitation, is the primary physiological driver for establishing legume–rhizobium symbiosis, this topic has been comprehensively reviewed elsewhere. Here, we focus specifically on environmental stresses that disrupt symbiosis after its initiation, including drought, salinity, flooding, temperature extremes, nutrient imbalance, oxidative stress, and chemical contaminants such as heavy metals, hydrocarbons, and pesticides. We examine how these stresses regulate symbiotic signaling, infection processes, oxygen homeostasis, nitrogenase protection, phytohormonal regulation, antioxidant defenses, exopolysaccharide production, and plasmid-mediated adaptation that collectively determine nodulation efficiency and biological nitrogen fixation. The review further discusses the roles of root nodule-associated microorganisms and microbial interactions in maintaining symbiotic stability under adverse environmental conditions. Finally, recent advances in multi-omics, CRISPR-based engineering, synthetic biology, nanotechnology, and stress-resilient microbial consortia are highlighted as promising approaches for developing next-generation bioinoculants and climate-resilient, sustainable agricultural systems.

2. Stress Responsive Reprogramming of Symbiotic Signaling Networks

A number of developmental and physiological processes, such as rhizobial survival in the rhizosphere, flavonoid-mediated signaling, nod factor perception, infection thread formation, nodule organogenesis, bacteroid differentiation, and biological nitrogen fixation, must be coordinated in order to establish and maintain legume–rhizobium symbiosis [18]. This coordination is disrupted by environmental stressors at almost every stage of the symbiotic process. Salinity and drought reduce rhizobial growth, change root exudation patterns, and inhibit flavonoid synthesis, which lowers the emergence of infection threads and Nod factor signaling. While heavy metals, hydrocarbons, and pesticides cause oxidative damage, membrane malfunction, and metabolic suppression in both symbiotic partners, flooding and soil compaction restrict the amount of oxygen available. In order to maintain symbiotic signaling, nodule function, and nitrogen fixation under harsh environmental conditions, stress resilience depends on integrated regulatory networks functioning in both the host plant and rhizobia [19]. These disruptions ultimately decrease nodulation efficiency, nitrogenase activity, and plant nitrogen acquisition [20]. These adaptive mechanisms and their functions in preserving symbiotic performance under stress are discussed in the following sections.

2.1. Environmental Regulation of Nod Factor Signaling and Symbiotic Regulatory Hubs

The molecular dialogue between legumes and rhizobia, bridging genera such as Azorhizobium, Bradyrhizobium, Mesorhizobium, Ensifer (Sinorhizobium), and Rhizobium relies on an intricate signaling cascade that initiates symbiotic nitrogen fixation and is dynamically shaped by environmental stress [21] (Figure 1). The primary stress driving plants to form beneficial microbial associations is nutrient stress (deficiency), particularly nitrogen and/or phosphorus limitation in the soil [22,23]. Nitrogen (N) and phosphorus (P) availability are major regulators of legume–rhizobium symbiosis, influencing nodule formation and biological nitrogen fixation [22,24]. The plant’s assessment of internal nutritional stress (N and P deprivation) does not occur in isolation; rather, it shares an intricate molecular crosstalk with physical and chemical environmental pressures such as drought, salinity and xenobiotics [25]. Under high nitrate conditions, NIN-LIKE PROTEINs (NLP1 and NLP2) suppress nodulation by inhibiting the master regulator NODULE INCEPTION (NIN) [26,27]. Simultaneously, nitrate induces root-derived CLE peptides, which are perceived by shoot receptor kinases such as SUNN or NARK, activating the Autoregulation of Nodulation (AON) pathway to systemically inhibit further nodule formation [28,29]. Conversely, nitrogen deficiency promotes nodulation through the CEP–CRA2–miR2111 signaling module, in which root-derived CEP peptides activate shoot-localized CRA2 receptors, resulting in the production of mobile miR2111 that suppresses the nodulation inhibitor TOO MUCH NODULATION (TML) in roots [29]. Nevertheless, phosphorus availability acts as a metabolic checkpoint because nitrogen fixation requires large amounts of ATP. Under phosphate starvation, the transcription factor PHR1 represses NIN expression, limits photosynthate allocation to nodules, and suppresses biological nitrogen fixation until sufficient phosphate becomes available [3].

The establishment and maintenance of effective symbiotic nitrogen fixation in rhizobia depend on a highly coordinated network of stress-responsive regulatory hubs, notably the NodD, RpoN–NifA, and FixLJ systems (Figure 1) [30]. These nodes function as a dynamic, hierarchical signaling group that integrates environmental, metabolic, and host-derived cues to ensure that the demanding process of nitrogen fixation is initiated only under optimal conditions. At the center of symbiotic signal transduction are the GRAS-family transcription factors NSP1 and NSP2, which function as a heterodimer downstream of microbial Nod and Myc factor perception [31]. Calcium-dependent signaling activates the NSP1–NSP2 complex, enabling NSP1 to induce the expression of early symbiotic genes, including NIN, while NSP2 stabilizes the complex through its conserved leucine heptad repeat domain [32,33]. Beyond activating nodulation, the NSP1–NSP2 complex regulates rhizosphere chemistry by promoting the biosynthesis of signaling metabolites such as flavonoids and strigolactones through interactions with transcription factors including MYB40, thereby enhancing rhizobial attraction and symbiotic establishment [31,34]. However, abiotic stresses such as drought and salinity interfere with this regulatory network. Stress conditions promote the accumulation of DELLA proteins, which interact with NSP2 and redirect transcriptional activity toward stress adaptation rather than symbiosis. Concurrently, abscisic acid (ABA) suppresses calcium spiking required for early symbiotic signaling, while excessive reactive oxygen species (ROS) oxidize leghemoglobin, increasing oxygen availability within nodules and impairing the oxygen-sensitive nitrogenase enzyme, ultimately reducing nitrogen fixation [22,35].

Root-exuded flavonoids activate the LysR-type regulator nodD, inducing core nod genes (nodABC, nodIJ) responsible for lipochitooligosaccharide Nod factor biosynthesis through binding to conserved nod box sequences [36]. While core genes encode conserved structural motifs, host-specific nod genes introduce modifications that confer species selectivity and compatibility. NodD isoforms expand their sensing beyond flavonoids and, in Rhizobium tropici CIAT 899, activate nodulation- and stress-related genes under high salinity or osmotic stress, independently of host signals. However, NodD activity is not just dependent on plant signals; it is also modulated by abiotic stress factors such as soil pH, salinity, and temperature, highlighting its role as an environmentally adaptable entry point for symbiosis [37]. This adaptability enables rhizobial strains to fine-tune Nod factor production according to local environmental constraints, thereby optimizing host recognition and infection efficiency across diverse ecological niches. Beyond initiating root hair curling, NodD activity is a prerequisite for biofilm establishment, which is required for successful infection thread penetration [38]. In saline environments, isoflavone secretion is severely altered in hosts such as soybean, thereby altering the expression patterns of nod genes via NodD [39]. Modern genomic analysis shows that species like R. leguminosarum utilize NodD2 to enhance competitive nodule colonization, suggesting that redundant NodD copies allow the bacteria to buffer against variable flavonoid concentrations in different soil types [40].

Upon successful infection and nodule formation, regulatory control shifts toward oxygen and nutrient-sensitive systems, mainly the FixLJ two-component system and the RpoN–NifA regulatory module. The FixLJ system serves as a critical microoxic sensor, enabling rhizobia to detect the low-oxygen environment within the nodule. FixL, a membrane-associated sensor kinase containing a heme-PAS domain, undergoes conformational changes in the FG loop and beta strands in response to oxygen availability [41]. Under aerobic conditions, oxygen binding suppresses its kinase activity, whereas microoxic conditions trigger autophosphorylation and subsequent activation of the response regulator FixJ. Activated FixJ induces the expression of downstream targets, including nifA and fixK, as well as a broader regulon involved in specialized respiration and metabolic adaptation [42]. Transcriptomic studies further reveal that the FixLJ regulon extends beyond nitrogen fixation genes to include pathways supporting redox balance and energy metabolism under microoxic stress [43]. In Mesorhizobium ciceri, FixLJ-FixK works in tandem with FnrN to regulate nitrogen fixation. This overlapping control ensures that the nif and fix genes are not activated early until a stable microoxic environment is achieved within the nodule [44]. FnrN is inactive under free living microaerobic conditions and becomes active from the interzone II–III stage, where it is essential for fixNOQP expression and nitrogen fixation. The hFixL–FxkR–FixK pathway indirectly supports this process by inducing fnrN under microaerobic conditions, priming it for activation in the nitrogen fixing zone [45].

The RpoN–NifA system functions as the terminal regulatory checkpoint controlling nitrogen fixation by directly activating nitrogenase (nif) and associated genes [46]. NifA acts as an enhancer-binding protein that works with the alternative sigma factor RpoN (σ⁵⁴) to initiate transcription, but its activity is extremely sensitive to oxygen due to conserved cysteine residues that inactivate the protein even at trace O₂ levels, thereby preventing wasteful expression of oxygen-sensitive nitrogenase [47]. This regulation operates within a hierarchical oxygen-sensing cascade in which hFixL and FnrN respond in sequence to declining oxygen levels, eventually allowing NifA to activate nifHDK expression under near-anoxic conditions [45]. Beyond oxygen control, NifA also regulates genes involved in molybdenum and iron acquisition, electron transport, and redox balance, ensuring proper assembly and function of the nitrogenase complex under nutrient limitation [47]. Recent studies further reveal that NifA integrates nitrogen fixation with broader symbiotic and stress-related processes, including phytohormone regulation. In β-rhizobia such as Paraburkholderia phymatum, NifA represses auxin biosynthesis genes (iaaMH) to prevent hypernodulation and conserve energy, whereas in α-rhizobia it can positively regulate hormone production, such as gibberellins in Bradyrhizobium diazoefficiens, contributing to nodule development [48]. Regulation of nifA itself varies across species, being either independent or controlled by oxygen-responsive systems such as FixLJ or alternative hFixL–FxkR pathways that interact with FnrN, forming feedback loops for fine-tuned expression [49]. Together, these multilayered controls highlight NifA as a central hub linking oxygen sensing, metabolism, and symbiotic efficiency.

These regulatory systems do not operate in isolation but are closely interconnected, forming a spatiotemporal relay that reflects the progression from rhizospheric signaling to intracellular nitrogen fixation [45]. NodD mediated signaling establishes the initial symbiotic framework, FixLJ responds to the declining oxygen gradient during nodule development, and RpoN–NifA executes the final activation of nitrogen fixation under near-anoxic conditions. Host-derived factors such as leghaemoglobins, sulfate transporters, and nutrient exchange proteins further reinforce this integration by maintaining the microoxic environment and supporting metabolic exchange [50]. This multilayered regulatory architecture functions as a reliable mechanism, ensuring that nitrogen fixation proceeds only when host signals, oxygen limitation, and cellular metabolic capacity are optimally aligned.

2.2. Stress-Driven Modulation of Alternative Infection Route

Environmental stresses frequently impair canonical Nod factor signaling (Figure 2), infection thread development, and symbiotic establishment by altering flavonoid secretion, redox balance, membrane signaling, and host immune responses [4]. Under such conditions, rhizobia can employ alternative infection mechanisms that enhance symbiotic adaptability, among which the Type III Secretion System (T3SS) plays a central role [51]. The T3SS is a syringe-like molecular complex composed of ~20–25 proteins that inject effector proteins directly into host plant cells, thereby modulating host responses [52]. Among the seven known T3SS families in Gram-negative bacteria, rhizobia predominantly utilize the α-RhcI type for interactions with legumes [52]. In most rhizobia, expression of the ‘tts’ region genes and associated Type III effectors (T3Es) depends on flavonoids and the coordinated action of NodD and TtsI. Flavonoid-activated NodD binds to nod box sequences to induce ttsI, after which TtsI activates transcription of T3SS structural and effector genes via tts box promoters [53]. These gene clusters are typically located near symbiotic loci such as nod-genes [52]. However, regulatory variation exists in the β-rhizobium, Cupriavidus taiwanensis, where T3SS expression is independent of flavonoids and TtsI and is instead induced by glutamate, resembling regulation in plant pathogens such as Ralstonia solanacearum and Pseudomonas aeruginosa [54]. Although α- and β-rhizobia share conserved symbiotic genes, including nod, nif, and fix, they differ in the regulatory hubs that coordinate symbiotic establishment. In α-rhizobia, such as Sinorhizobium, Bradyrhizobium, and Mesorhizobium, flavonoid-activated NodD functions as the central regulator linking host signal perception to Nod factor biosynthesis and secretion-system activation, whereas β-rhizobia such as Cupriavidus taiwanensis can regulate the T3SS independently of flavonoids, NodD, and TtsI, responding instead to environmental and metabolic signals such as glutamate. These differences contribute to variation in host specificity and symbiotic efficiency, indicating that α-rhizobia rely more strongly on host-derived flavonoid signalling, whereas β-rhizobia integrate broader environmental cues into symbiotic regulation [55,56,57]. In α-rhizobia, ttsI is usually the only gene preceded by a nod box, underscoring its central regulatory role. Moreover, in M. loti R7A, the Type IV secretion system (T4SS) regulator VirA is also controlled by NodD and flavonoids, indicating that different secretion systems can be integrated into a common symbiotic regulatory framework [54].

Under stress conditions, host plants often exhibit enhanced defense signaling involving ROS, ethylene, salicylic acid, jasmonic acid, and MAP kinase pathways, which can suppress rhizobial colonization and nodulation [58]. T3SS effectors function as adaptive regulators by suppressing host immune responses to facilitate colonization while simultaneously determining host specificity. In compatible hosts, these effectors promote infection, whereas in incompatible genotypes they can be recognized by plant resistance (R) genes, triggering effector-triggered incompatibility (ETI) and blocking nodulation [51]. In genera such as Bradyrhizobium and Ensifer, this duality is particularly evident. In some Vigna species, certain strains (e.g., BTAi1 and ORS285) can bypass Nod factor signaling completely and rely on T3SS-mediated infection, where effectors act as primary signals targeting downstream symbiotic pathways or modulating plant hormone signaling, such as cytokinin pathways, to induce nodulation [59]. Such Nod factor-independent infection strategies may provide adaptive flexibility when environmental stress disrupts conventional symbiotic signaling [60]. Effectors like NopL suppress host defense by interfering with MAP kinase signaling [61]. However, this system can also be detrimental, as host plants carrying specific R genes (e.g., Rj2 or Rfg1 in soybean) may recognize effectors such as NopP or NopL and initiate hypersensitive responses that prevent symbiosis [62] (Table 1).

Recent studies have demonstrated that Type VI secretion system (T6SS)’s role in legume–rhizobium symbiosis extends beyond antagonism to include host colonization, symbiotic establishment, and rhizosphere adaptation [63,64]. Although the Type III secretion system (T3SS) has historically received greater attention in rhizobial symbiosis, recent studies have highlighted the importance of T6SS in regulating beneficial plant–microbe interactions [64,65]. Ensifer fredii USDA257 harbors both T3SS and a Group 3 T6SS which, despite lacking antibacterial activity in vitro, is essential for efficient root colonization, nodulation, nitrogen fixation, and competitive occupancy of soybean nodules [64,66]. Its expression is induced under nutrient limitation and during the stationary phase, suggesting an adaptive role in rhizosphere persistence [64]. Similar roles have been reported in Rhizobium etli Mim1, where T6SS and its effector Re78 enhance symbiotic competitiveness [67,68] and in Paraburkholderia phymatum, where T6SS-b is activated during early symbiosis and regulated by σ54 and cyclic di-GMP signalling [69,70]. Rhizobial microsymbionts associated with Oxytropis spp. also possess both T3SS and T6SS that contribute to efficient nodule development and symbiotic effectiveness [66]. T6SS also contributes to beneficial plant-microbe interactions in PGPR while enabling related species such as Paraburkholderia sabiae to suppress phytopathogens [65,71].

From an evolutionary perspective, T3SS genes are often located on symbiotic islands or plasmids, facilitating horizontal gene transfer and rapid diversification of symbiotic traits. Environmental stress and rhizosphere instability can further promote genomic flexibility and selection for alternative infection strategies [72]. Similar to T3SS, T4SS also deliver effector molecules into host cells and can either enhance or inhibit symbiosis depending on the host context, further highlighting the complexity and adaptive plasticity of rhizobial–plant interactions under fluctuating environmental conditions [52]. Despite their significant mechanistic potential, the agronomic application of T3SS-, T4SS-, and T6SS-mediated infection pathways remains limited by their strong dependence on rhizobial strain, host genotype, and environmental conditions [64,65]. While these secretion systems can enhance nodulation, rhizosphere competitiveness, and stress adaptation, they may also trigger host immune responses or exhibit inconsistent performance under field conditions due to competition with indigenous microbiota and variable soil environments [73,74]. Therefore, their successful exploitation in sustainable agriculture will require careful host–strain selection, multi-location field validation, and ecological risk assessment before they can be incorporated into next-generation rhizobial inoculants [64,72].

Table 1. Effects of Rhizobial T3SS Effectors on Legume-Rhizobium Interactions.

T3E	Rhizobial Strain	Host Plant	Positive Effect	Negative Effect	Ref.
Bel2-5	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Triggers NF-independent nodulation	Restricts nodule formation	[74]
		Rj4 soybean
ErnA	Bradyrhizobium sp. ORS3257	Aeschynomene indica	Triggers NF-independent nodulation and cell division		[61]
	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Positively affects nodulation		[74]
GunA/GunA2	B. diazoefficiens USDA110	Cajanus cajan	Promotes nodulation		[75]
	Ensifer fredii HH103	Soybean cultivars	Promotes nodulation in some cultivars	Negative effects in some soybean interactions	[76]
InnB	Bradyrhizobium elkanii USDA61	Vigna mungo	Promotes nodulation		[77]
		Vigna cv. KPS1		Restricts nodulation
		Vigna angularis			[78]
		Soybean cv. BARC-2 (Rj4)	Promotes nodulation		[74]
NopAA	Ensifer fredii HH103	Glycine max	Promotes rhizobial infection		[77]
		Vigna unguiculata cv. Red Caloona		Negatively affects symbiosis
NopAB	Bradyrhizobium sp. ORS3257	Vigna mungo	Promotes nodule formation		[61,77]
		Vigna unguiculata
		Aeschynomene indica	Minor positive role		[61]
NopAO	Bradyrhizobium sp. ORS3257	Aeschynomene indica		Negatively affects symbiosis	[78]
NopC	Ensifer fredii HH103	Soybean	Positively affects nodulation		[79]
		Vigna unguiculata
		Lotus japonicus GIFU		Blocks nodulation
NopD	Bradyrhizobium sp. XS1105	Tephrosia vogelii		Suppresses nodule formation	[80]
	E. fredii HH103	Glycine max	Positive regulation in some germplasms	Negative regulation in some germplasms	[81]
NopE	B. diazoefficiens USDA110	Glycine max	Promotes nodulation		[82]
		Macroptilium atropurpureum
		Vigna radiata cv. KPS1
		Vigna radiata cv. KPS2		Negatively affects nodulation via SA-mediated ETI
NopF	Bradyrhizobium elkanii USDA61	Lotus spp.		Inhibits infection and nodulation	[83]
NopJ	Rhizobium sp. NGR234	Crotalaria juncea		Minor negative effect on nodulation	[84]
		Lablab purpureus		Negative effect on nodulation	[72]
NopL	E. fredii NGR234	Phaseolus vulgaris	Inhibits nodule senescence; promotes nodulation		[85]
		Flemingia congesta	Promotes nodulation		[86]
	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Positively affects nodulation		[74]
NopM	Rhizobium sp. NGR234	Lablab purpureus	Promotes nodulation		[87]
		Lotus japonicus		Reduces nodule formation	[88]
	Bradyrhizobium sp. ORS3257	Aeschynomene indica	Positively promotes nodulation		[61]
	Bradyrhizobium elkanii USDA61	Lotus spp.		Induces ETI-like response and early nodule senescence	[84]
NopP	Rhizobium sp. NGR234	Flemingia congesta	Promotes nodulation		[72]
		Tephrosia vogelii
		Vigna unguiculata		Inhibits nodulation
	Bradyrhizobium diazoefficiens USDA110	Soybean carrying GmNNL1		Inhibits root hair infection	[74]
	Bradyrhizobium diazoefficiens USDA122	Rj2 soybean		Severely restricts nodulation via ETI-like response	[62]
	E. fredii HH103	Glycine max	Positive regulation in some germplasms	Negative regulation in some germplasms	[89]
	Mesorhizobium amorphae	Robinia pseudoacacia	Promotes nitrogen-fixing activity and nodule biomass		[90]
NopP2	B. vignae ORS3257	Vigna mungo	Promotes nodulation		[77]
	Bradyrhizobium elkanii USDA61				[91]
NopT	Rhizobium sp. NGR234	Phaseolus vulgaris	Positively affects nodulation		[92]
		Tephrosia vogelii
		Crotalaria juncea		Suppresses nodulation	[84]
		Crotalaria pallida
	Ensifer fredii USDA257	Glycine max cv. Nenfeng 15		Impairs nodulation	[93]
	Ensifer fredii HH103	Soybean germplasms	Positive/neutral effects in some germplasms	Negative effects in others	[94]
	Mesorhizobium amphore CCNWGS0123	Robinia pseudoacacia	Minor positive effect on nodulation		[95]
	Bradyrhizobium sp. ORS3257	Vigna mungo	Promotes nodule formation		[77]
		Vigna unguiculata			[61]
		Aeschynomene indica	Lesser positive role
NopX	Rhizobium sp. NGR234	Lablab purpureus	Promotes symbiosis		[72]
		Pachyrhizus tuberosus
		Flemingia congesta

Table 1. Effects of Rhizobial T3SS Effectors on Legume-Rhizobium Interactions.

T3E	Rhizobial Strain	Host Plant	Positive Effect	Negative Effect	Ref.
Bel2-5	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Triggers NF-independent nodulation	Restricts nodule formation	[74]
		Rj4 soybean
ErnA	Bradyrhizobium sp. ORS3257	Aeschynomene indica	Triggers NF-independent nodulation and cell division		[61]
	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Positively affects nodulation		[74]
GunA/GunA2	B. diazoefficiens USDA110	Cajanus cajan	Promotes nodulation		[75]
	Ensifer fredii HH103	Soybean cultivars	Promotes nodulation in some cultivars	Negative effects in some soybean interactions	[76]
InnB	Bradyrhizobium elkanii USDA61	Vigna mungo	Promotes nodulation		[77]
		Vigna cv. KPS1		Restricts nodulation
		Vigna angularis			[78]
		Soybean cv. BARC-2 (Rj4)	Promotes nodulation		[74]
NopAA	Ensifer fredii HH103	Glycine max	Promotes rhizobial infection		[77]
		Vigna unguiculata cv. Red Caloona		Negatively affects symbiosis
NopAB	Bradyrhizobium sp. ORS3257	Vigna mungo	Promotes nodule formation		[61,77]
		Vigna unguiculata
		Aeschynomene indica	Minor positive role		[61]
NopAO	Bradyrhizobium sp. ORS3257	Aeschynomene indica		Negatively affects symbiosis	[78]
NopC	Ensifer fredii HH103	Soybean	Positively affects nodulation		[79]
		Vigna unguiculata
		Lotus japonicus GIFU		Blocks nodulation
NopD	Bradyrhizobium sp. XS1105	Tephrosia vogelii		Suppresses nodule formation	[80]
	E. fredii HH103	Glycine max	Positive regulation in some germplasms	Negative regulation in some germplasms	[81]
NopE	B. diazoefficiens USDA110	Glycine max	Promotes nodulation		[82]
		Macroptilium atropurpureum
		Vigna radiata cv. KPS1
		Vigna radiata cv. KPS2		Negatively affects nodulation via SA-mediated ETI
NopF	Bradyrhizobium elkanii USDA61	Lotus spp.		Inhibits infection and nodulation	[83]
NopJ	Rhizobium sp. NGR234	Crotalaria juncea		Minor negative effect on nodulation	[84]
		Lablab purpureus		Negative effect on nodulation	[72]
NopL	E. fredii NGR234	Phaseolus vulgaris	Inhibits nodule senescence; promotes nodulation		[85]
		Flemingia congesta	Promotes nodulation		[86]
	Bradyrhizobium elkanii USDA61	nfr1 mutant soybean	Positively affects nodulation		[74]
NopM	Rhizobium sp. NGR234	Lablab purpureus	Promotes nodulation		[87]
		Lotus japonicus		Reduces nodule formation	[88]
	Bradyrhizobium sp. ORS3257	Aeschynomene indica	Positively promotes nodulation		[61]
	Bradyrhizobium elkanii USDA61	Lotus spp.		Induces ETI-like response and early nodule senescence	[84]
NopP	Rhizobium sp. NGR234	Flemingia congesta	Promotes nodulation		[72]
		Tephrosia vogelii
		Vigna unguiculata		Inhibits nodulation
	Bradyrhizobium diazoefficiens USDA110	Soybean carrying GmNNL1		Inhibits root hair infection	[74]
	Bradyrhizobium diazoefficiens USDA122	Rj2 soybean		Severely restricts nodulation via ETI-like response	[62]
	E. fredii HH103	Glycine max	Positive regulation in some germplasms	Negative regulation in some germplasms	[89]
	Mesorhizobium amorphae	Robinia pseudoacacia	Promotes nitrogen-fixing activity and nodule biomass		[90]
NopP2	B. vignae ORS3257	Vigna mungo	Promotes nodulation		[77]
	Bradyrhizobium elkanii USDA61				[91]
NopT	Rhizobium sp. NGR234	Phaseolus vulgaris	Positively affects nodulation		[92]
		Tephrosia vogelii
		Crotalaria juncea		Suppresses nodulation	[84]
		Crotalaria pallida
	Ensifer fredii USDA257	Glycine max cv. Nenfeng 15		Impairs nodulation	[93]
	Ensifer fredii HH103	Soybean germplasms	Positive/neutral effects in some germplasms	Negative effects in others	[94]
	Mesorhizobium amphore CCNWGS0123	Robinia pseudoacacia	Minor positive effect on nodulation		[95]
	Bradyrhizobium sp. ORS3257	Vigna mungo	Promotes nodule formation		[77]
		Vigna unguiculata			[61]
		Aeschynomene indica	Lesser positive role
NopX	Rhizobium sp. NGR234	Lablab purpureus	Promotes symbiosis		[72]
		Pachyrhizus tuberosus
		Flemingia congesta

2.3. Integration of Abiotic Stress Signals with Host Compatibility

Successful legume rhizobium symbiosis depends on the plant’s ability to integrate microbial recognition with environmental stress perception. Under optimal conditions, rhizobial Nod factors are recognized by LysM-type receptor kinases such as NFR1/NFR5 in Lotus japonicus and LYK3/NFP in Medicago truncatula, initiating a conserved symbiotic signaling cascade that includes calcium oscillations decoded by CCaMK and downstream transcriptional regulators such as CYCLOPS, NSP1, NSP2, and NIN [96]. These signaling events coordinate root hair curling, infection thread formation, and cortical cell division necessary for nodule organogenesis. However, abiotic stresses such as salinity, drought, and elevated temperature can interfere with this recognition system by altering membrane stability, receptor conformation, and ligand-binding affinity. Stress-induced perturbations in plasma membrane fluidity may reduce the sensitivity of Nod factor receptors, thereby weakening signal perception and downstream symbiotic responses. In L. japonicus, the plasma membrane-localized receptors NFR1, NFR5, and NFRe function as the primary recognition complex for Nod factor signaling, with receptor-associated kinases such as NiCK4 linking perception to intracellular signaling pathways [97]. DELLA proteins further stabilize this signaling axis under abiotic stress, reinforcing symbiotic signaling even under unfavorable environmental conditions [98].

Beyond Nod factor recognition, legumes employ an additional compatibility barrier to authenticate bacterial identity before allowing intracellular colonization. This double-check system ensures that only compatible rhizobia can establish infection threads and persist in symbiosis during stress [99]. Exopolysaccharide (EPS) perception represents a critical secondary surveillance mechanism operating downstream of Nod factor signaling [100]. In L. japonicus, the receptor-like kinase Exopolysaccharide Receptor 3 (EPR3) directly binds bacterial EPS and discriminates between compatible and incompatible rhizobial strains [101]. EPR3 belongs to the LysM receptor kinase family and is transcriptionally induced by Nod factor signaling in root hairs and epidermal cells. Structurally, EPR3 contains an active intracellular kinase domain and is proposed to function together with a co-receptor containing an inactive pseudokinase domain for efficient recognition of microbial surface carbohydrates [102]. This layered recognition strategy highlights that successful nodulation is not solely dependent on Nod factor perception but also requires continuous monitoring of rhizobial surface compatibility during infection progression (Figure 1). EPS also contribute to infection thread stability, osmotic protection, and mitigation of oxidative stress caused by salinity and drought [103]. Auxin and cytokinin signaling further integrate with these recognition systems to regulate nodule organogenesis and infection thread development, emphasizing the coordinated nature of host-microbe communication.

Abiotic stress signaling also intersects with symbiotic regulation through hormonal gating mechanisms, particularly involving abscisic acid (ABA). Under stress conditions, ABA levels rise, shifting the plant’s physiological priorities from growth and symbiosis toward survival and water conservation. Elevated ABA can suppress Nod factor-induced calcium spiking, inhibit infection thread formation, and downregulate nodulation-associated genes, effectively preventing the establishment of energetically expensive symbioses under unfavorable conditions [104]. In this context, ABA functions as a molecular “gatekeeper,” allowing the plant to evaluate whether sufficient resources are available to sustain nitrogen-fixing nodules. Ethylene signaling further interacts with ABA and auxin pathways, forming an interconnected hormonal network that modulates compatibility decisions during stress exposure. Rather than functioning through isolated signaling pathways, nodulation resilience emerges through dynamic integration of stress perception, receptor-mediated microbial recognition, and hormonal crosstalk (Figure 3). This systems-level coordination enables legumes to balance defense, metabolic cost, and symbiotic benefit, thereby maintaining selective and stress-resilient host compatibility.

3. Genetic and Regulatory Determinants of Rhizobial Stress Adaptation

Adaptive responses cannot rely on a single regulatory mechanism since environmental stresses target numerous stages of symbiosis simultaneously. Instead, transcriptional regulators, signaling molecules, protective metabolites, respiratory adaptations, and microbial interactions function in coordination to maintain nodulation and nitrogen fixation, which leads to effective stress tolerance [105]. The following sections discuss these interconnected regulatory networks.

3.1. Salinity and Drought-Responsive Mechanisms in Rhizobia

Salinity affects nearly 20% of the world’s cultivated land and nearly 50% of irrigated agricultural land making it one of the major constraints to global crop production [106]. Rhizobia from stressed environments possess specialized physiological and genetic adaptations that enhance stress tolerance (Table 2). Fast-growing species such as E. fredii and E. meliloti generally perform better under saline conditions than slow-growing rhizobia such as Bradyrhizobium spp. and R. leguminosarum [93,107]. A major adaptation involves the accumulation of compatible solutes, including trehalose, proline, glutamate, and NAGGN, which stabilize osmotic balance and protein integrity under stress [108]. In E. meliloti, salt tolerance is mediated by stress-responsive genes homologous to Escherichia coli osmotic stress systems, including kdp, trkA/H, and aqp genes involved in potassium transport and osmotic regulation [109]. The strain Rm1021 also contains aqpZ1 and aqpZ2, potentially associated with glycerol transport. Long-term osmotic adaptation relies on uptake and biosynthesis of osmoprotectants such as trehalose and glycine betaine through BCCT transporters and genes including treAB, treY, treS, treZ, proU, proP, and betT. Glycine betaine synthesis from choline is mediated by the betICBA operon, while plant-derived choline induces betI expression and may enhance nitrogen fixation in Medicago spp. [110].

Stress adaptation and symbiotic efficiency are further regulated by the stringent response mediated by guanosine tetra- and penta-phosphate (ppGpp), synthesized by RelA/SpoT (Rsh) homologs [111]. ppGpp regulates Nod factor and EPS production, iron homeostasis, oxidative stress responses, and T3SS activity. Mutations in relA significantly impair nodulation and infection efficiency in Medicago hosts [112], while stress-associated genes such as typA contribute to host-dependent symbiotic performance [113]. EPS-producing rhizobia further enhance stress resilience by improving root adhesion, water retention, soil aggregation, and ion sequestration under saline conditions [114]. For example, R. azibense SR-26 improves ionic balance and antioxidant activity in host plants, reducing Na⁺ accumulation and preserving chlorophyll stability under salinity stress [115].

Drought similarly compromises symbiotic nitrogen fixation by disrupting carbon allocation and nodule metabolism. Legumes respond by accumulating antioxidants such as superoxide dismutase, catalase, glutathione, and ascorbate, along with osmoprotectants that maintain cellular hydration and redox balance [116]. Nodulated legumes often exhibit a “sink-priority” strategy that preferentially allocates resources to roots and nodules while restricting vegetative growth [117]. In soybean, nodules maintain strong sink activity and continue receiving photosynthates during moderate drought, although severe stress reduces carbon flow, causing sucrose accumulation and decreased malate production, ultimately limiting nitrogen fixation [117]. Enhanced drought tolerance in nodulated plants is associated with osmotic adjustment, regulation of aquaporins, and coordinated transcriptional responses involving MYB, NAC, NF-Y, bHLH, and GRAS transcription factors [118]. Rhizobial genes such as otsA, Auc, and Usp contribute to osmoprotection and drought tolerance in M. ciceri Ca181 [119], while strain-dependent antioxidant and compatible solute accumulation improve drought resilience in faba bean [120]. Additional regulatory mechanisms include salicylic acid-mediated ROS signaling via A4-rolB, which enhances flavonol accumulation and activates stress-responsive transcription factors such as DREBs and HSFs [121]. In M. truncatula, drought resilience is also strengthened through coordinated regulation of miRNAs, lncRNAs, and mRNAs, particularly the miR169l-centered ceRNA network that activates MtNF-YA2 and MtNF-YA3 and sustains drought-responsive gene expression [122].

Table 2. Key genetic regulators mediating abiotic stress tolerance and symbiotic fitness in rhizobia.

Gene/Molecule	Function in Stress Adaptation	Rhizobia	Evidence	In Vivo/In Vitro	Refs.
otsA–otsB	Trehalose biosynthesis; protects proteins and membranes during osmotic, salt and desiccation stress; improves nodulation under salinity.	Mesorhizobium sp. CCBAU25338; B. diazoefficiens 110spc4	Arachis hypogaea; Glycine max	In vivo	[123,124]
treY–treZ	Alternative trehalose biosynthetic pathway contributing to osmoprotection and desiccation tolerance.	Mesorhizobium cicero Rch125	Cicer arietinum	In vivo	[125]
treS	Trehalose–maltose interconversion; carbon homeostasis and osmoprotection.	Bradyrhizobium japonicum USDA 110	Glycine max	In vivo	[126]
betS	High-affinity glycine betaine transporter maintaining osmotic balance under salt stress.	Bradyrhizobium japonicum USDA110	-	In vitro	[127]
relA	ppGpp synthesis; stringent response controlling adaptation to nutrient starvation and stress while supporting symbiotic competence.	Rhizobium etli CFN42	-	In vitro	[128]
spoT	Maintains ppGpp homeostasis during nutritional stress.
typA (bipA)	Ribosome-associated GTPase regulating stress-responsive translation and efficient nodulation.	Ensifer meliloti 1021	Medicago truncatula	In vivo	[129]
rpoH1	Heat-shock sigma factor controlling molecular chaperones and oxidative stress genes.	Ensifer meliloti BY294	Medicago sativa	In field	[130]
rpoE2	Extracytoplasmic sigma factor mediating envelope, osmotic and oxidative stress responses.	Ensifer meliloti Rm1021	Medicago sativa, Medicago truncatula	In vivo	[131]
groEL–groES	Chaperonin complex promoting protein folding under heat and oxidative stress; overexpression improves symbiotic effectiveness.	Ensifer meliloti groESL	Medicago sativa	In vivo	[132]
dnaK–dnaJ	Heat-shock chaperones preventing protein aggregation during thermal stress.	Rhizobium tropici CIAT899	Phaseolus vulgaris	In vivo	[133]
clpB	ATP-dependent protein disaggregase required for recovery from heat stress.	Mesorhizobium ciceri LMS-1	Cicer arietinum	In vivo	[134]
katG	Catalase-peroxidase detoxifying hydrogen peroxide generated during infection and stress.	Rhizobium etli	Phaseolus vulgaris	In vivo	[135]
katE	Catalase protecting against oxidative damage.	R. leguminosarum	Pisum sativum	In vivo	[136]
sodA/sodB	Superoxide dismutases scavenging reactive oxygen species.	Mesorhizobium huakuii 7653R	Astragalus sinicus	In vivo	[137]
oxyR	Global oxidative stress regulator activating antioxidant defence genes.	Rhizobium etli	Phaseolus vulgaris	In vivo	[135]
exoY, exoA, exoF	Exopolysaccharide biosynthesis essential for biofilm formation, osmoprotection and infection thread development.	Mesorhizobium japonicum R7A	Lotus japonicus	In vivo	[138]
ndvA/ndvB	Cyclic β-glucan biosynthesis required for osmotic adaptation and successful infection.	Rhizobium (Ensifer) sp. NGR234	Leucaena leucocephala, Vigna unguiculata	In vivo	[139]
bacA	Membrane protein protecting bacteroids against host NCR peptides and ensuring persistence within nodules.	Rhizobium leguminosarum 3841	Phaseolus vulgaris	In vivo	[140]
fixL–fixJ	Two-component oxygen-sensing system activating microaerobic metabolism and nitrogen fixation genes.	Bradyrhizobium japonicum strains 110spc4, 9043, and 9039K2	Glycine max	In vivo	[141]
fixK	Downstream transcription factor regulating the low-oxygen regulon.
nifA	Master activator of nitrogen fixation genes under microaerobic conditions.	Rhizobium leguminosarum UPM791	Pisum sativum	In vivo	[142]
nodD	Flavonoid-responsive regulator initiating nod gene expression under environmental cues.	Rhizobium meliloti MPIK3030	Medicago sativa	In vivo	[143]
nolR	Global repressor modulating nod gene expression to optimize host adaptation.	Ensifer fredii HH103-1	Vigna unguiculata	In vitro	[144]
phoB/phoR	Phosphate-starvation response improving adaptation to phosphorus limitation and influencing symbiosis.	Agrobacterium tumefaciens C58	-	In vitro	[145]
chvG/chvI	Acid-responsive two-component regulatory system maintaining envelope integrity under low pH.	Rhizobium leguminosarum VF39	Pisum sativum, Vicia nigricans, Lens culinaris	In vivo	[146]
actA/actR	Acid tolerance determinants maintaining intracellular pH homeostasis.	Ensifer meliloti WSM419	-	In vitro	[147]

Table 2. Key genetic regulators mediating abiotic stress tolerance and symbiotic fitness in rhizobia.

Gene/Molecule	Function in Stress Adaptation	Rhizobia	Evidence	In Vivo/In Vitro	Refs.
otsA–otsB	Trehalose biosynthesis; protects proteins and membranes during osmotic, salt and desiccation stress; improves nodulation under salinity.	Mesorhizobium sp. CCBAU25338; B. diazoefficiens 110spc4	Arachis hypogaea; Glycine max	In vivo	[123,124]
treY–treZ	Alternative trehalose biosynthetic pathway contributing to osmoprotection and desiccation tolerance.	Mesorhizobium cicero Rch125	Cicer arietinum	In vivo	[125]
treS	Trehalose–maltose interconversion; carbon homeostasis and osmoprotection.	Bradyrhizobium japonicum USDA 110	Glycine max	In vivo	[126]
betS	High-affinity glycine betaine transporter maintaining osmotic balance under salt stress.	Bradyrhizobium japonicum USDA110	-	In vitro	[127]
relA	ppGpp synthesis; stringent response controlling adaptation to nutrient starvation and stress while supporting symbiotic competence.	Rhizobium etli CFN42	-	In vitro	[128]
spoT	Maintains ppGpp homeostasis during nutritional stress.
typA (bipA)	Ribosome-associated GTPase regulating stress-responsive translation and efficient nodulation.	Ensifer meliloti 1021	Medicago truncatula	In vivo	[129]
rpoH1	Heat-shock sigma factor controlling molecular chaperones and oxidative stress genes.	Ensifer meliloti BY294	Medicago sativa	In field	[130]
rpoE2	Extracytoplasmic sigma factor mediating envelope, osmotic and oxidative stress responses.	Ensifer meliloti Rm1021	Medicago sativa, Medicago truncatula	In vivo	[131]
groEL–groES	Chaperonin complex promoting protein folding under heat and oxidative stress; overexpression improves symbiotic effectiveness.	Ensifer meliloti groESL	Medicago sativa	In vivo	[132]
dnaK–dnaJ	Heat-shock chaperones preventing protein aggregation during thermal stress.	Rhizobium tropici CIAT899	Phaseolus vulgaris	In vivo	[133]
clpB	ATP-dependent protein disaggregase required for recovery from heat stress.	Mesorhizobium ciceri LMS-1	Cicer arietinum	In vivo	[134]
katG	Catalase-peroxidase detoxifying hydrogen peroxide generated during infection and stress.	Rhizobium etli	Phaseolus vulgaris	In vivo	[135]
katE	Catalase protecting against oxidative damage.	R. leguminosarum	Pisum sativum	In vivo	[136]
sodA/sodB	Superoxide dismutases scavenging reactive oxygen species.	Mesorhizobium huakuii 7653R	Astragalus sinicus	In vivo	[137]
oxyR	Global oxidative stress regulator activating antioxidant defence genes.	Rhizobium etli	Phaseolus vulgaris	In vivo	[135]
exoY, exoA, exoF	Exopolysaccharide biosynthesis essential for biofilm formation, osmoprotection and infection thread development.	Mesorhizobium japonicum R7A	Lotus japonicus	In vivo	[138]
ndvA/ndvB	Cyclic β-glucan biosynthesis required for osmotic adaptation and successful infection.	Rhizobium (Ensifer) sp. NGR234	Leucaena leucocephala, Vigna unguiculata	In vivo	[139]
bacA	Membrane protein protecting bacteroids against host NCR peptides and ensuring persistence within nodules.	Rhizobium leguminosarum 3841	Phaseolus vulgaris	In vivo	[140]
fixL–fixJ	Two-component oxygen-sensing system activating microaerobic metabolism and nitrogen fixation genes.	Bradyrhizobium japonicum strains 110spc4, 9043, and 9039K2	Glycine max	In vivo	[141]
fixK	Downstream transcription factor regulating the low-oxygen regulon.
nifA	Master activator of nitrogen fixation genes under microaerobic conditions.	Rhizobium leguminosarum UPM791	Pisum sativum	In vivo	[142]
nodD	Flavonoid-responsive regulator initiating nod gene expression under environmental cues.	Rhizobium meliloti MPIK3030	Medicago sativa	In vivo	[143]
nolR	Global repressor modulating nod gene expression to optimize host adaptation.	Ensifer fredii HH103-1	Vigna unguiculata	In vitro	[144]
phoB/phoR	Phosphate-starvation response improving adaptation to phosphorus limitation and influencing symbiosis.	Agrobacterium tumefaciens C58	-	In vitro	[145]
chvG/chvI	Acid-responsive two-component regulatory system maintaining envelope integrity under low pH.	Rhizobium leguminosarum VF39	Pisum sativum, Vicia nigricans, Lens culinaris	In vivo	[146]
actA/actR	Acid tolerance determinants maintaining intracellular pH homeostasis.	Ensifer meliloti WSM419	-	In vitro	[147]

3.2. Detoxification and Xenobiotic Resistance Pathways in Rhizobia Under Stress

Legumes growing in metal-contaminated soils often establish symbiosis with metal-tolerant rhizobia and, in some cases, arbuscular mycorrhizal fungi, which collectively contribute to nutrient acquisition and metal stress tolerance [148]. Rhizobial tolerance mechanisms include metallothioneins, metal efflux systems such as czcABC, antioxidant enzymes, hormone regulation, and membrane-stabilization pathways that reduce metal-induced oxidative damage [116]. Metal-contaminated environments frequently select for tolerant genera such as Cupriavidus, while species including B. japonicum and E. meliloti enhance tolerance to arsenic and multiple metals, including aluminum, copper, iron, and molybdenum [149]. Certain strains, including B. pachyrhizi and R. selenitireducens, combine nitrogen fixation with metal detoxification functions [150]. In Medicago truncatula, reduced arsenic accumulation in shoots correlates with altered expression of the nitrate transporter gene NRT3.1-like under abscisic acid regulation [151]. Similarly, B. canariense L-7AH maintains effective symbiosis with Lupinus albus under elevated mercury concentrations without major impairment of photosynthesis or nitrogen fixation [152], while E. medicae enhances mercuric reductase activity that converts toxic Hg²⁺ into elemental mercury. Rhizobium induced m6A RNA methylation in soybean under cadmium stress further indicates epigenetic regulation of metal tolerance [153].

EPS contribute substantially to metal detoxification by binding, sequestering, or precipitating toxic ions, thereby reducing their bioavailability and oxidative effects [154]. EPS-rich biofilms function as protective matrices in the rhizosphere, while nodules themselves act as structural barriers through lignification, phenolic accumulation, and redox regulation [155]. Rhizobium sp. HGR-4 enhances nickel tolerance by immobilizing nickel within nodules while maintaining nitrogen fixation and leghaemoglobin production [156]. Additional molecular mechanisms include arsenic methylation, glutathione-mediated ROS buffering, thiol-based chelation, and metal efflux systems [157]. In R. leguminosarum, glutathione contributes to intracellular cadmium sequestration [158], whereas arsenite-oxidizing strains such as Rhizobium sp. NT-26 convert As(III) into the less toxic As(V) form [159]. Metal-tolerant strains including Cupriavidus STM 6070 possess multiple heavy metal resistance genes and RND-type transport systems associated with resistance to nickel, zinc, and copper [160]. Similarly, R. pusense CR02 reduces Cr(VI) through chromate reductases, EPS-mediated sequestration, and antioxidant activity [161].

Functional studies further demonstrate that copper and zinc resistance genes, such as cueO, yedYZ, and cusA-like loci, are important for maintaining nodulation and plant growth under metal stress [162]. In Ensifer meliloti, transporters including ZntA (Zn²⁺ ATPase) and CopA1b (Cu⁺ ATPase) contribute to metal tolerance and metalloenzyme function [163]. Under heavy metal stress, legumes additionally exhibit structural modifications in nodules, including cell wall thickening, altered vacuolation, phenolic accumulation, and reduced infection thread formation, as observed in Anthyllis vulneraria [164].

Rhizobia contribute to bioremediation by metabolizing inorganic and organic contaminants in agricultural and industrial environments. E. meliloti and E. medicae efficiently removed nitrates from sugarcane effluents, with removal efficiency influenced by inoculum density and exposure duration. Several species of Rhizobium, Mesorhizobium, and related genera also degrade persistent pesticides and agrochemicals [165]. Ensifer sp. W16 degraded the herbicide fomesafen while maintaining nitrogen fixation in soybean [166], whereas Rhizobium sp. MRP1 tolerated multiple pesticides and responded with increased EPS production, suggesting contaminant immobilization functions [167]. Petroleum hydrocarbons, particularly PAHs and alkanes, adversely affect rhizobial symbiosis by reducing nodulation, nitrogenase activity, and biomass accumulation [168].

Rhizobial adaptation to xenobiotic-contaminated environments involves mechanisms that preserve symbiotic functioning and stress tolerance under toxic conditions. In hydrocarbon- and metal-contaminated soils, rhizobia experience oxidative stress, membrane damage, and impaired nodulation. To maintain symbiosis, they employ integrated responses including EPS production, biofilm formation, redox regulation, and metal sequestration [169] (Table 3).

Genomic analyses indicate that rhizobia inhabiting contaminated environments possess broad metabolic adaptability linked to stress resilience. Rhizobia, such as R. pusense, survive in hydrocarbon-rich rhizospheres through pathways for the utilization of long-chain hydrocarbons and aromatic compounds [170]. Neorhizobium phenanthreniclasticum Rsf11 has been studied to exhibit nickel resistance mediated through EPS-dependent adsorption and metal efflux systems [171], while R. petrolearium SL-1 persists in phenanthrene-contaminated environments through flexible aromatic-compound metabolism [172].

Such metabolic and physiological adaptations may support bioremediation by enabling rhizobia to persist within contaminated rhizospheres, sustain root colonization, and maintain nitrogen-fixing interactions under toxic stress conditions [41]. EPS-rich biofilms additionally reduce contaminant bioavailability, buffer oxidative stress, and stabilize rhizosphere microenvironments, thereby improving microbial survival and plant performance in polluted soils [173]. Collectively, these adaptive pathways link xenobiotic tolerance with maintenance of symbiotic stability, highlighting the ecological role of rhizobia as stress-resilient partners in contaminated agroecosystems.

EPS supports xenobiotic degradation by promoting the biofilm formation, microbial attachment, and stabilization of degradative enzymes, such as laccases and peroxidases [174]. EPS functional groups additionally bind toxic compounds and reduce oxidative and hydraulic stress. Devosia polycyclovorans Naph2^T degrades high-molecular-weight PAHs, including pyrene and benzo[a]pyrene, through a specialized pca gene cluster linked to TCA cycle intermediates [175].

3.3. Genomic Plasticity and Horizontal Gene Transfer in Stress Adaptation

Plasmids are major components of rhizobial genomes that facilitate adaptation between free-living soil conditions and nitrogen-fixing symbiosis. These mobile extrachromosomal elements carry essential symbiotic genes, including nod, nif, and fix, which regulate nodulation and nitrogen fixation, while also contributing to metabolic flexibility, stress tolerance, and ecological adaptation [176]. Horizontal gene transfer (HGT), recombination, and genome rearrangements mediated by plasmids drive rhizobial diversification and host adaptation, although plasmid instability may generate ineffective non-symbiotic strains. While many lineages of rhizobia carry their essential symbiosis genes on distinct, easily transmissible plasmids, the evolutionary paradigms of both Mesorhizobium and Bradyrhizobium break from this convention [177,178]. Rather than utilizing symbiotic plasmids, these genera harbour their nodulation and nitrogen-fixation genes directly within their chromosomes inside specialized mobile genetic elements traditionally referred to as symbiosis islands. Deeper genomic deconstructions reveal that these islands are more accurately categorized as highly dynamic, active symbiotic Integrative Conjugative Elements (symICEs) [179,180]. These elements are capable of site-specific integration at precise tRNA genes and can adopt complex monopartite or polypartite (bipartite and tripartite) configurations, driving large-scale chromosomal rearrangements and allowing critical symbiotic traits to rapidly transfer and reshuffle across diverse bacterial populations [180,181].

In E. meliloti, the megaplasmids pSymA and pSymB function as distinct yet complementary genomic compartments (Figure 4). pSymA primarily contains nod, nif, and fix gene clusters required for host infection and nitrogen fixation, together with genes associated with nitrate reduction, nutrient metabolism, secretion systems, and stress adaptation within the rhizosphere [182]. In contrast, pSymB acts as a metabolic chromid carrying housekeeping genes involved in cell division, carbohydrate metabolism, vitamin biosynthesis, tRNA synthesis, and surface polysaccharide production. Many osmoresponsive genes linked to salt and osmotic tolerance are concentrated within pSymB regions [183].

Accessory and non-symbiotic plasmids further enhance rhizobial genomic plasticity by encoding traits associated with complex carbon utilization, antibiotic resistance, heavy-metal tolerance, and heat-stress adaptation. Comparative genomic studies indicate that although plasmid size and organization vary considerably, symbiotic plasmids retain conserved nod and nif/fix gene clusters essential for symbiosis [184]. Many plasmids additionally display mosaic evolutionary origins in which symbiotic genes derive from distinct ancestral lineages, contributing to host specificity and ecological diversification.

Integration of acquired symbiotic genes into existing regulatory and metabolic networks involves genome remodeling, transposable element activity, and regulation by global regulators such as MucR, which links symbiosis with nutrient acquisition and stress responses [185]. Environmental adaptation is further influenced by HGT-driven dissemination of adaptive loci such as the nickel resistance nre operon among rhizobia inhabiting serpentine soils [186].

Mobile genomic elements contribute to rhizobial ecological diversification beyond classical legume symbiosis. In R. moroccanus sp. nov., accessory plasmids and genomic islands encode nutrient-acquisition systems, stress-response pathways, nitrogen-fixation genes, and biosynthetic clusters associated with adaptation to alkaline dryland soils and endophytic colonization of non-legume hosts [187]. Despite lacking canonical nod genes, the strain retains nitrogen-fixing capacity and secondary metabolite production, indicating specialization toward a plant-associated lifestyle. Accessory plasmids may also carry genes such as hrrP, which encodes a metallopeptidase that degrades host NCR peptides and enhances resistance to host-imposed differentiation and antimicrobial stress [188].

HGT also facilitates the dissemination of stress-adaptive loci, including heavy metal resistance (czc), hydrocarbon degradation (alkB), and error-prone DNA polymerases linked to accelerated stress adaptation [189]. Prophages activated under oxidative or chemical stress further promote genomic exchange through transduction, mobilizing genes involved in metal detoxification, antioxidant defense (sod, katG), hydrocarbon degradation, and EPS biosynthesis [190]. EPS-rich biofilms additionally act as hotspots for phage-mediated gene transfer and genomic diversification [191].

Figure 4. Circular maps of representative rhizobial megaplasmids showing the distribution of genes associated with nodulation, nitrogen fixation, stress tolerance, transport, secretion systems, and metabolic adaptation relevant to plant-microbe interactions [192,193,194,195].

Figure 4. Circular maps of representative rhizobial megaplasmids showing the distribution of genes associated with nodulation, nitrogen fixation, stress tolerance, transport, secretion systems, and metabolic adaptation relevant to plant-microbe interactions [192,193,194,195].

Table 3. Rhizobial strategies for enhancing various stress tolerances.

Stress	Microorganism	Plant Species	Function	Ref.
Drought	Glomus intraradices and native rhizobial strains	Genotypes of Phaseolus vulgaris	Positive correlation between mycorrhizal colonization and nodule trehalose content	[196]
	Ensifer sp.	Acacia senegal	Increased EPS production and protection of Ensifer cell cultures	[197]
	Rhizobium tropici and Paenibacillus polymyxa	Phaseolus vulgaris	Upregulation of stress tolerance genes	[198]
	Rhizophagus irregularis and Mesorhizobium tianshanense	Glycyrrhiza uralensis	Improved phosphorus nutrition	[199]
	Bradyrhizobium liaoningense and Ambispora leptoticha	Glycine max	Improved yield parameters	[200]
Heavy Metal Stress (Ni, Co, Fe, Cr, As)	Rhizobium leguminosarum and Mesorhizobium septentrionale	Lotus corniculatus	Accumulation of phenols and remodeling of apoplast in nodules	[96]
	Ensifer meliloti	Medicago sativa	Disruption of aqpS arsenite transport/aquaglyceroporin gene	[201]
	Bradyrhizobium pachyrhizi and Ochrobactrum anthropic	Pongamia pinnata	Increased nitrogen and leghaemoglobin content; EPS production; metal detoxification through accumulation, precipitation, methylation, and chelation	[150]
	Rhizobium selenitireducens and Rhizobium pisi	Macrotyloma uniflorum	Enhanced metal stress tolerance	[156]
	Rhizobium sp.	—	Heavy metal tolerance mechanisms	[202]
	Rhizobium sp.	—	Metal detoxification and stress adaptation	[157]
	Rhizobium HGR-4, HGR-6, HGR-13, and HGR-25	Macrotyloma uniflorum	Enhanced nitrogen and leghaemoglobin content	[156]
	Ensifer meliloti 1021, Rm5038, and Rm5055	Medicago truncatula	Inhibition of ABA and linalool biosynthesis in roots	[151]
Salinity	Rhizobium azibense	Phaseolus vulgaris (L.)	Increased antioxidant defense enzymes	[115]
	Rhizobia sp. RhLO1, Glomus sp., Sclerocystis sp., and Acaulospora sp.	Medicago sativa L.	Higher PPO and POX activity	[203]
	Native rhizobia	Medicago truncatula and Phaseolus vulgaris	Enhanced trehalose accumulation	[204]
	Rhizobium leguminosarum and arbuscular mycorrhizal fungi	Vicia faba	Increased catalase, peroxidase, SOD, and proline accumulation	[205]
	Alcaligenes faecalis MH-2, Alcaligenes sp. MH-3, Achromobacter denitrificans MH-6, and Sphingobacterium spiritivorum MH-9	Cicer arietinum (L.)	Enhanced nutrient absorption and antioxidant responses	[206]
Hydrocarbon Stress	Neorhizobium petrolearium and Rhizobium phenanthrenilyticum	Medicago sativa L.	Degradation of phenanthrene, fluorene, and anthracene	[112]
	Mesorhizobium cicer sp. LGR33	Cicer arietinum (L.)	Improved pesticide tolerance and bio-inoculant formulation	[207]
Pesticides	Bradyrhizobium japonicum	Vigna radiata (L.)	Increased nodulation and symbiotic efficiency	[208]
	Bradyrhizobium sp.	Vigna radiata (L.)	Increased productivity and reduced glyphosate and quizalofop toxicity	[209]
	Mesorhizobium ciceri BRM5	Cicer arietinum (L.)	Enhanced productivity under pesticide stress	[210]

Table 3. Rhizobial strategies for enhancing various stress tolerances.

Stress	Microorganism	Plant Species	Function	Ref.
Drought	Glomus intraradices and native rhizobial strains	Genotypes of Phaseolus vulgaris	Positive correlation between mycorrhizal colonization and nodule trehalose content	[196]
	Ensifer sp.	Acacia senegal	Increased EPS production and protection of Ensifer cell cultures	[197]
	Rhizobium tropici and Paenibacillus polymyxa	Phaseolus vulgaris	Upregulation of stress tolerance genes	[198]
	Rhizophagus irregularis and Mesorhizobium tianshanense	Glycyrrhiza uralensis	Improved phosphorus nutrition	[199]
	Bradyrhizobium liaoningense and Ambispora leptoticha	Glycine max	Improved yield parameters	[200]
Heavy Metal Stress (Ni, Co, Fe, Cr, As)	Rhizobium leguminosarum and Mesorhizobium septentrionale	Lotus corniculatus	Accumulation of phenols and remodeling of apoplast in nodules	[96]
	Ensifer meliloti	Medicago sativa	Disruption of aqpS arsenite transport/aquaglyceroporin gene	[201]
	Bradyrhizobium pachyrhizi and Ochrobactrum anthropic	Pongamia pinnata	Increased nitrogen and leghaemoglobin content; EPS production; metal detoxification through accumulation, precipitation, methylation, and chelation	[150]
	Rhizobium selenitireducens and Rhizobium pisi	Macrotyloma uniflorum	Enhanced metal stress tolerance	[156]
	Rhizobium sp.	—	Heavy metal tolerance mechanisms	[202]
	Rhizobium sp.	—	Metal detoxification and stress adaptation	[157]
	Rhizobium HGR-4, HGR-6, HGR-13, and HGR-25	Macrotyloma uniflorum	Enhanced nitrogen and leghaemoglobin content	[156]
	Ensifer meliloti 1021, Rm5038, and Rm5055	Medicago truncatula	Inhibition of ABA and linalool biosynthesis in roots	[151]
Salinity	Rhizobium azibense	Phaseolus vulgaris (L.)	Increased antioxidant defense enzymes	[115]
	Rhizobia sp. RhLO1, Glomus sp., Sclerocystis sp., and Acaulospora sp.	Medicago sativa L.	Higher PPO and POX activity	[203]
	Native rhizobia	Medicago truncatula and Phaseolus vulgaris	Enhanced trehalose accumulation	[204]
	Rhizobium leguminosarum and arbuscular mycorrhizal fungi	Vicia faba	Increased catalase, peroxidase, SOD, and proline accumulation	[205]
	Alcaligenes faecalis MH-2, Alcaligenes sp. MH-3, Achromobacter denitrificans MH-6, and Sphingobacterium spiritivorum MH-9	Cicer arietinum (L.)	Enhanced nutrient absorption and antioxidant responses	[206]
Hydrocarbon Stress	Neorhizobium petrolearium and Rhizobium phenanthrenilyticum	Medicago sativa L.	Degradation of phenanthrene, fluorene, and anthracene	[112]
	Mesorhizobium cicer sp. LGR33	Cicer arietinum (L.)	Improved pesticide tolerance and bio-inoculant formulation	[207]
Pesticides	Bradyrhizobium japonicum	Vigna radiata (L.)	Increased nodulation and symbiotic efficiency	[208]
	Bradyrhizobium sp.	Vigna radiata (L.)	Increased productivity and reduced glyphosate and quizalofop toxicity	[209]
	Mesorhizobium ciceri BRM5	Cicer arietinum (L.)	Enhanced productivity under pesticide stress	[210]

4. Exopolysaccharides as Structural and Signaling Determinants of Symbiotic Resilience

Rhizobial cells produce a wide range of surface-associated polysaccharides, including extracellular polysaccharides (EPS), lipopolysaccharides, capsular polysaccharides, cyclic β-glucans, K-antigen polysaccharides, gel-forming polysaccharides, and cellulose fibrils [211]. Among these, EPS are the most extensively studied because of their central roles in host recognition, infection thread (IT) development, stress tolerance, and biofilm formation. EPS are structurally diverse heteropolymers composed primarily of hexose and uronic acid residues, often modified by succinate, pyruvate, and acetate groups that determine their physicochemical properties and biological functions (Table 4). As major constituents of the extracellular polymeric matrix, EPS interact with proteins, lipids, and extracellular nucleic acids derived from microbial metabolism and cell lysis, forming a hydrated protective matrix around rhizobial cells [191]. Structural diversity in EPS, including variations in sugar composition, glycosidic linkages, polymer length, and non-carbohydrate substitutions, serves as an important molecular determinant of host specificity and symbiotic compatibility, particularly in indeterminate nodule-forming legumes associated with Ensifer (Sinorhizobium) meliloti and Rhizobium leguminosarum [103]. In E. meliloti, two major EPS species are produced: succinoglycan (EPS I), consisting of octasaccharide repeating units containing one galactose and seven glucose residues, and galactoglucan (EPS II), composed of alternating glucose and galactose residues linked by α-1,3 and β-1,3 glycosidic bonds [103]. Both polymers occur as high- and low-molecular-weight fractions, although only the low-molecular-weight forms are biologically active during symbiosis. Functional modifications such as succinylation, acetylation, and pyruvylation are essential for infection thread initiation and elongation, whereas mutations in EPS biosynthetic genes, including pssA and exoB in R. leguminosarum, result in defective infection and ineffective nodules. Likewise, Mesorhizobium loti synthesizes an O-acetylated EPS specifically recognized by the Lotus receptor EPR3, highlighting the importance of EPS structural integrity during host recognition [101].

Comprehensive characterization of rhizobial exopolysaccharides (EPS) relies on complementary analytical techniques. Monosaccharide composition is commonly determined using gas chromatography-mass spectrometry (GC–MS), high-performance liquid chromatography (HPLC), or high-performance anion-exchange chromatography (HPAEC) following acid hydrolysis [212,213]. Fourier transform infrared (FTIR) spectroscopy is routinely employed to identify characteristic functional groups, including hydroxyl, carboxyl, acetyl, pyruvyl, and succinyl moieties, thereby providing rapid confirmation of polysaccharide composition [214]. Molecular mass, repeating-unit composition, and substitution patterns are further resolved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), electrospray ionization mass spectrometry (ESI-MS), and tandem MS (MS/MS) [213]. Complete structural elucidation, including glycosidic linkage positions, branching patterns, anomeric configurations, and non-carbohydrate substitutions, primarily relies on one- and two-dimensional nuclear magnetic resonance (¹H, ¹³C, COSY, TOCSY, HSQC, HMBC, and NOESY) spectroscopy, often combined with methylation linkage analysis and size-exclusion chromatography [215,216]. X-ray diffraction (XRD) spectroscopy is commonly used to analyze the structures, such as crystallinity or amorphousness, of the EPS [212]. These complementary approaches have enabled detailed structural characterization of EPS from model symbionts such as Ensifer meliloti, Rhizobium leguminosarum, Rhizobium tropici, and Mesorhizobium loti. However, despite the rapid expansion of rhizobial genome sequences, experimentally validated EPS structural data remain available for only a limited number of representative strains, and for many recently described species or environmental isolates, EPS composition is inferred from biosynthetic gene clusters rather than direct biochemical characterization, highlighting an important gap in our understanding of EPS diversity and its relationship with host specificity and environmental adaptation [177,214,217].

EPS biosynthesis is tightly coordinated by environmental cues and host-derived signals to optimize both symbiotic efficiency and adaptation to adverse conditions. In E. meliloti, phosphate limitation activates the PhoR/PhoB regulatory system, stimulating EPS II production, whereas the quorum-sensing regulators SinI/SinR/ExpR induce EPS biosynthesis and activate the glycanase ExsH, which converts high-molecular-weight succinoglycan into the symbiotically active low-molecular-weight form [103]. Global regulators such as MucR and the ExoS/ChvI two-component system promote the transition from motile to sessile growth by repressing flagellar biosynthesis while enhancing EPS production, root colonization, and biofilm development. This lifestyle transition is further regulated by cyclic di-GMP (c-di-GMP), whose elevated intracellular concentration stimulates EPS synthesis and biofilm formation while suppressing bacterial motility. Plant-derived flavonoids also modulate these regulatory networks; for example, NodD1 in Ensifer fredii HH103 represses EPS production while simultaneously activating Nod factor biosynthesis and the Type III secretion system [103]. Environmental stresses substantially influence these signaling pathways. Salinity reduces diguanylate cyclase activity and succinoglycan synthesis, thereby weakening biofilm formation and root hair colonization [39]. Acidic conditions impair polymerization proteins such as ExoL and ExoP or activate the ExoS/ChvI stress-response pathway, collectively reducing EPS production. Phosphate deficiency additionally shifts EPS synthesis from succinoglycan toward galactoglucan, altering biofilm architecture and symbiotic performance. Beyond their structural functions, EPS contribute significantly to heavy metal tolerance through functional groups including carboxyl, hydroxyl, amino, phosphate, and sulfate moieties that facilitate biosorption, ion exchange, chelation, and metal precipitation [159]. Together, these properties enable EPS to improve water retention, nutrient acquisition, surface attachment, osmotic buffering, and microbial persistence under adverse environmental conditions.

The protective functions of EPS become particularly critical during infection thread development, where maintenance of structural integrity is essential for successful bacterial invasion and nodule formation, especially under drought and other abiotic stresses. Infection threads are specialized plant cell wall invaginations whose elongation is highly sensitive to dehydration-induced mechanical instability, potentially resulting in thread deformation or arrest of bacterial transport [218]. Rhizobial EPS forms a hydrated capsule surrounding bacterial cells during IT formation, facilitating bacterial movement through host tissues while simultaneously protecting both partners from osmotic stress. In determinate nodule-forming legumes such as soybean and Lotus japonicus, EPS is indispensable for root hair curling, infection thread formation, bacterial release into host cells, bacteroid differentiation, and efficient nodulation [219]. Progression of infection threads depends on continuous molecular communication between rhizobia and the host, extending beyond initial Nod factor recognition by the NFR1 and NFR5 receptors. In L. japonicus, this second layer of quality control is mediated by the LysM receptor kinase EPR3, whose expression is induced following Nod factor signaling to evaluate the structural compatibility of rhizobial EPS [101]. The EPR3 ectodomain possesses two atypical carbohydrate-binding domains (M1 and M2) together with a canonical LysM3 domain, enabling highly specific recognition of compatible EPS structures while discriminating against truncated or defective polymers with micromolar affinity [102,220]. Genetic studies have demonstrated that epr3 mutants exhibit impaired infection thread progression despite normal Nod factor perception, confirming that EPS recognition functions as a second checkpoint following symbiotic initiation. Recent evidence further indicates that the constitutively expressed receptor EPR3a provides baseline EPS surveillance, whereas inducible EPR3 reinforces compatibility assessment during infection. Together, these receptors promote infection thread advancement in response to compatible EPS while restricting colonization by defective or incompatible rhizobia [100,101,221]. This two-tiered recognition mechanism, whereby Nod factors license bacterial entry and EPS validates surface integrity, represents an evolutionarily conserved strategy that optimizes host specificity and symbiotic fidelity across nitrogen-fixing plants [222]. Although EPR3 displays ligand promiscuity by recognizing EPS monomers from Mesorhizobium, Rhizobium, and Ensifer, it remains highly selective against chitin and lipochitooligosaccharides, thereby maintaining strict surveillance of compatible symbionts [102]. The evolutionary significance of this pathway is underscored by pseudogenization of the EPR receptor in Parasponia, suggesting that EPS perception played a major role during symbiont switching and host adaptation [222].

Successful infection thread development also depends on coordinated host and bacterial genetic responses that preserve cellular architecture under osmotic and mechanical stress. The host activates Rhizobium-directed Polar Growth (RPG), which coordinates subcellular reorganization during IT elongation, while the vesicle trafficking proteins VAMP721e and VAMP721d facilitate continuous deposition of membrane and cell wall material required for thread extension [218]. Simultaneously, the SCAR/WAVE cytoskeletal complex, including NAP1 and PIR1, stabilizes actin organization to prevent infection thread fragmentation [218]. In pea, overexpression of the LysM receptor PsLYK10 enhances activation of essential symbiotic regulators, including DNF2, NAD1, RSD, and SYMCRK, which suppress defense responses and promote successful bacteroid differentiation [220]. The nitrate transporter NIP/LATD functions as an additional developmental checkpoint by determining whether infection threads continue growing or are terminated through defense-associated responses such as suberin deposition [174]. Rhizobial regulatory proteins likewise contribute to infection success; mutations in the Ros/MucR-family transcriptional regulator RosR impair infection thread development, bacterial release, and bacteroid differentiation [223]. Collectively, EPS-mediated protection, receptor-based surveillance, and coordinated host-microbe genetic regulation establishes a robust symbiotic interface that maintains infection thread stability, preserves nodule homeostasis, and enhances the resilience of legume–rhizobium symbiosis under environmental stress.

Table 4. Comprehensive classification of microbial exopolysaccharides (EPS), their composition and functions.

Classification	EPS Type/ Example	Major Components/Structure	Primary Functions	Ref.
Homopolysaccharides	α-D-Glucans (Dextran, Mutan, Alternan, Reuteran)	Glucose polymers linked by α-glycosidic bonds	Biofilm formation, adhesion, protection from desiccation	[224,225,226]
	β-D-Glucans (Cellulose, Curdlan, Scleroglucan)	β-linked glucose polymers	Structural support, water retention, biofilm stability	[225,227,228]
	Fructans (Levan, Inulin)	Fructose polymers	Osmoprotection, carbon storage, antioxidant and prebiotic activities	[213,226,229]
	Polygalactans/Pullulan	Galactose or maltotriose polymers	Film formation, encapsulation, moisture retention	[225,227,230]
Heteropolysaccharides	Xanthan	Glucose, mannose, glucuronic acid	Biofilm formation, viscosity enhancement, emulsification	[225,231,232]
	Alginate	Mannuronic and guluronic acids	Water retention, metal chelation, biofilm matrix	[225,231,233]
	Gellan	Glucose, glucuronic acid, rhamnose	Gel formation and immobilization	[224,234]
	Kefiran	Glucose and galactose	Immunomodulatory, antimicrobial and probiotic effects	[235]
	Hyaluronic acid	Glucuronic acid and N-acetylglucosamine	Hydration, lubrication, tissue repair	[231,236]
Functional EPS	Constructive EPS	Neutral structural polymers	Biofilm scaffold, water retention, mechanical stability	[227,237,238]
	Sorptive EPS	Charged polymers	Metal chelation, nutrient adsorption, pollutant binding	[227,237]
	Surface-active EPS	Amphiphilic polymers	Adhesion, emulsification, antimicrobial activity	[227]
	Redox-active EPS	Electron-transfer capable EPS	Bioremediation, metal reduction, ROS modulation	[227,238]
	Informative EPS	Signaling polysaccharides	Cell–cell communication and host recognition	[227,237]
	Active EPS	Bioactive polymers	Antioxidant, antimicrobial and immunomodulatory activities	[227,237,238]
	Nutritive EPS	Storage polymers	Carbon and energy reserve	[227]
Symbiosis-specific EPS	EPS I (Succinoglycan)	Octasaccharide composed of glucose and galactose with succinyl, acetyl and pyruvyl substitutions	Infection thread development, immune suppression, biofilm formation	[101,102,239,240]
	EPS II (Galactoglucan)	Glucose–galactose repeating disaccharides	Biofilm formation and surface attachment	[240,241]
	Low-molecular-weight EPS	Short oligomers derived from EPS I or EPS II	Mobile signaling molecules	[101,102]
	High-molecular-weight EPS	Long-chain extracellular polymers	Biofilm matrix and hydration	[102,240]
	Cyclic β-(1→2)-glucans	Cyclic glucose polymers	Osmoregulation and membrane stability	[240]
	Capsular polysaccharides (KPS/CPS)	Surface-associated polysaccharides	Protection from environmental stress and phages	[240]

Table 4. Comprehensive classification of microbial exopolysaccharides (EPS), their composition and functions.

Classification	EPS Type/ Example	Major Components/Structure	Primary Functions	Ref.
Homopolysaccharides	α-D-Glucans (Dextran, Mutan, Alternan, Reuteran)	Glucose polymers linked by α-glycosidic bonds	Biofilm formation, adhesion, protection from desiccation	[224,225,226]
	β-D-Glucans (Cellulose, Curdlan, Scleroglucan)	β-linked glucose polymers	Structural support, water retention, biofilm stability	[225,227,228]
	Fructans (Levan, Inulin)	Fructose polymers	Osmoprotection, carbon storage, antioxidant and prebiotic activities	[213,226,229]
	Polygalactans/Pullulan	Galactose or maltotriose polymers	Film formation, encapsulation, moisture retention	[225,227,230]
Heteropolysaccharides	Xanthan	Glucose, mannose, glucuronic acid	Biofilm formation, viscosity enhancement, emulsification	[225,231,232]
	Alginate	Mannuronic and guluronic acids	Water retention, metal chelation, biofilm matrix	[225,231,233]
	Gellan	Glucose, glucuronic acid, rhamnose	Gel formation and immobilization	[224,234]
	Kefiran	Glucose and galactose	Immunomodulatory, antimicrobial and probiotic effects	[235]
	Hyaluronic acid	Glucuronic acid and N-acetylglucosamine	Hydration, lubrication, tissue repair	[231,236]
Functional EPS	Constructive EPS	Neutral structural polymers	Biofilm scaffold, water retention, mechanical stability	[227,237,238]
	Sorptive EPS	Charged polymers	Metal chelation, nutrient adsorption, pollutant binding	[227,237]
	Surface-active EPS	Amphiphilic polymers	Adhesion, emulsification, antimicrobial activity	[227]
	Redox-active EPS	Electron-transfer capable EPS	Bioremediation, metal reduction, ROS modulation	[227,238]
	Informative EPS	Signaling polysaccharides	Cell–cell communication and host recognition	[227,237]
	Active EPS	Bioactive polymers	Antioxidant, antimicrobial and immunomodulatory activities	[227,237,238]
	Nutritive EPS	Storage polymers	Carbon and energy reserve	[227]
Symbiosis-specific EPS	EPS I (Succinoglycan)	Octasaccharide composed of glucose and galactose with succinyl, acetyl and pyruvyl substitutions	Infection thread development, immune suppression, biofilm formation	[101,102,239,240]
	EPS II (Galactoglucan)	Glucose–galactose repeating disaccharides	Biofilm formation and surface attachment	[240,241]
	Low-molecular-weight EPS	Short oligomers derived from EPS I or EPS II	Mobile signaling molecules	[101,102]
	High-molecular-weight EPS	Long-chain extracellular polymers	Biofilm matrix and hydration	[102,240]
	Cyclic β-(1→2)-glucans	Cyclic glucose polymers	Osmoregulation and membrane stability	[240]
	Capsular polysaccharides (KPS/CPS)	Surface-associated polysaccharides	Protection from environmental stress and phages	[240]

5. Multi-Microbial Synergy: Rhizobia–Endophyte Interactions

5.1. Metabolic Complementarity and Antioxidant Reinforcement

The root nodule microbiome is a dynamic ecosystem composed of rhizobia, non-rhizobial endophytes (NREs), and associated microorganisms linked through complex metabolic interactions [242]. Beyond serving as sites of biological nitrogen fixation, nodules function as metabolically active microenvironments enriched with signaling molecules, phytohormones, and specialized metabolites that influence microbial interactions and symbiotic stability. A key feature of this system is metabolic cross-feeding, in which metabolites are exchanged through unidirectional, bidirectional, or bidirectional interactions among microbial partners [243]. These exchanges connect rhizobia with NREs and contribute to nutrient cycling, redox balance, stress adaptation, and nitrogen fixation efficiency.

Experimental studies demonstrate strong metabolic compatibility among nodule-associated microorganisms. Co-cultivation of Bradyrhizobium with actinobacteria such as Streptomyces and Terrabacter improves substrate utilization and plant performance [244]. In several legumes, only a fraction of nodule-inhabitants are true nodulating rhizobia, whereas numerous α-, β-, and γ-proteobacteria colonize nodules through rhizobial infection threads [245]. Although these NREs cannot independently induce nodules, they frequently enhance symbiotic performance through phosphate and potassium solubilization, siderophore and exopolysaccharide production, indole-3-acetic acid synthesis, ACC deaminase activity, and pathogen suppression [246]. Consequently, co-inoculation often improves nodulation, nutrient acquisition, stress tolerance, and crop productivity [247].

NREs also contribute to nutrient mobilization and the regulation of oxidative stress within nodules. Phosphate-solubilizing bacteria release organic acids and phosphatases that convert insoluble phosphorus into bioavailable forms required for ATP-dependent nitrogen fixation [248]. Genera including Azotobacter, Bacillus, Burkholderia, Enterobacter, Flavobacterium, Rhizobium, and Serratia are associated with phosphorus availability and nitrogenase stability [245]. Many nodule-associated bacteria additionally mineralize organic nitrogen, phosphorus, and sulfur compounds, sustaining metabolic balance within the nodule environment [249]. To counter oxidative stress, NREs produce antioxidant enzymes, including superoxide dismutase, catalase, and peroxidases, which detoxify reactive oxygen species and maintain redox homeostasis. These antioxidant systems help protect bacteroids, stabilize membrane integrity, and sustain nitrogen fixation under adverse environmental conditions [250].

Microbial partners regulate nodulation and plant physiology through phytohormone production and stress-buffering mechanisms. Many nodule-associated bacteria, including Mesorhizobium and Pseudomonas, produce indole-3-acetic acid, cytokinins, and related compounds that promote root growth, infection thread formation, vascular differentiation, and nutrient uptake, thereby increasing carbon allocation to nodules [251]. Stress protection is further mediated through exopolysaccharides, antioxidant enzymes, osmoprotectants, and biofilm formation, which reduce oxidative stress, osmotic imbalance, oxygen fluctuations, and ion toxicity while maintaining nitrogenase activity [242]. Archaeal groups including Thaumarchaeota, Crenarchaeota, and Euryarchaeota, along with fungal endophytes and protists, additionally contribute to nitrogen cycling, nutrient exchange, microbial dispersal, and stress adaptation within nodules [252].

5.2. Hormonal Modulation Mediated Stress Alleviation

Phytohormones are central regulators of symbiotic development and nodulation, influencing both infection processes and the expression of common symbiosis pathway genes [253]. Among these, ethylene functions primarily as a negative regulator of nodulation. Application of ethylene or its precursor 1-aminocyclopropane-1-carboxylate (ACC) suppresses Ca²⁺ spiking, symbiotic gene expression, infection thread (IT) formation, and nodule organogenesis [254]. On the contrary, inhibition of ethylene biosynthesis using aminoethoxyvinylglycine (AVG) enhances IT formation and nodulation [255]. Ethylene-insensitive mutants such as Mtsickle in M. truncatula display hypernodulation phenotypes and altered transcriptional responses to Nod factor signaling, further confirming the inhibitory role of ethylene during early symbiosis [256,257]. Elevated ethylene production and increased expression of ethylene biosynthesis genes have also been reported in rhizobia-inoculated legumes [258].

Ethylene biosynthesis is regulated by the sequential conversion of methionine to S-adenosylmethionine (SAM), ACC, and finally ethylene, catalyzed by SAM synthase, ACC synthase (ACS), and ACC oxidase, respectively [259]. ACS is generally considered the rate-limiting enzyme controlling ethylene production [260]. Ethylene perception occurs through membrane-bound receptors including ETR1, ETR2, ERS1, ERS2, and EIN4, which negatively regulate signaling in the absence of ethylene through CTR1-mediated repression of EIN2 [261]. Ethylene binding inactivates CTR1, allowing EIN2 cleavage and activation of EIN3-dependent transcriptional responses [262]. Mutations in Ein2 or overexpression of dominant-negative ethylene receptor alleles result in enhanced nodulation and infection, underscoring the importance of ethylene signaling in restricting symbiotic development [263].

Microbial modulation of ethylene biosynthesis is a major adaptive mechanism that enhances nodulation under stress conditions. Many rhizobia and plant growth-promoting endophytes produce ACC deaminase, which cleaves ACC into α-ketobutyrate and ammonia, thereby reducing substrate availability for ethylene synthesis [264]. By lowering stress-induced ethylene accumulation, ACC deaminase-producing microbes alleviate ethylene-mediated inhibition of root elongation, infection thread formation, and nodulation under salinity, drought, and heavy metal stress [265]. Co-inoculation of legumes with rhizobia and ACC deaminase-producing non-rhizobial endophytes often results in enhanced nodulation, nitrogen fixation, and stress tolerance compared with single inoculations [16]. Importantly, these microbes fine-tune rather than completely suppress ethylene signaling, preserving its essential functions in defense and developmental regulation [266].

Nodulation is also governed by extensive phytohormonal crosstalk involving auxin, cytokinin, and abscisic acid (ABA). Auxin regulates root architecture and nodule primordia development [267], whereas cytokinin positively controls cortical cell division and nodule organogenesis [268]. ABA interacts with ethylene and other hormonal pathways to modulate nodulation under environmental stress [104]. Collectively, these findings demonstrate that nodulation is controlled through an integrated hormonal and microbial regulatory network that balances plant defense, stress adaptation, and symbiotic efficiency under shifting environmental conditions. Although rhizobia–endophyte co-inoculation has consistently enhanced nodulation, nitrogen fixation, nutrient acquisition, and crop productivity under controlled conditions, the stability of these synergistic interactions under field conditions remains variable [268]. Successful establishment of introduced microbial consortia depends on their ability to compete with indigenous rhizobia and resident soil microbiota for root colonization and nodule occupancy, while environmental factors such as soil type, moisture, temperature, nutrient availability, and crop genotype strongly influence inoculant persistence and efficacy [269]. Meta-analyses of field studies indicate that co-inoculation with rhizobia and plant growth-promoting bacteria generally improves nodulation, nitrogenase activity, nutrient uptake, and grain yield across several grain legumes, although the magnitude of these benefits varies considerably among locations and growing seasons [270,271]. Consistent yield improvements have been demonstrated in multi-site field trials using Bradyrhizobium spp. together with Azospirillum brasilense in soybean and cowpea, as well as Rhizobium–Bradyrhizobium co-inoculation in common bean and Pararhizobium giardinii, Ochrobactrum sp. co-inoculation with non-rhizobial endophytes Serratia spp. on lentil, supporting the agronomic potential of multi-microbial inoculants [272,273,274]. However, long-term, multi-location field evaluations remain necessary to identify competitive, environmentally adapted microbial consortia capable of maintaining stable symbiotic interactions under diverse agroecosystems.

6. Systemic Regulation of Nodulation Under Environmental Stress

6.1. Phytohormonal and Cle-Peptide Mediated Regulation of Stress-Responsive Nodulation

Phytohormones form a central regulatory axis for plant development and stress adaptation, coordinating root architecture, cellular differentiation, and defense signaling. Under abiotic and xenobiotic stresses such as salinity, drought, and hydrocarbon toxicity, plants activate a dynamic hormonal network involving auxins, cytokinins, gibberellins (GA), ethylene, abscisic acid (ABA), and secondary messengers like jasmonic acid (JA), brassinosteroids (BRs), salicylic acid (SA), and nitric oxide (NO) [275]. Rhizobia introduce an additional regulatory layer by producing phytohormones or modulating their metabolism, thereby reshaping plant responses when endogenous signaling is insufficient [276].

Rhizobial IAA biosynthesis, often driven by tryptophan in root exudates, stimulates root branching, biomass accumulation, and nodule initiation, creating a positive feedback loop with host auxin production [277]. Experimental inoculations with IAA-producing strains have consistently enhanced root elongation and shoot growth under stress [278]. Ethylene, by contrast, accumulates during stress and inhibits Nod factor signaling, thus constraining nodule initiation [279]. Rhizobia alleviate this constraint via ACC deaminase (acdS), which degrades the ethylene precursor, improving colonization and nodulation efficiency [280].

Other hormones contribute complementary roles. Gibberellin intermediates (e.g., GA9) synthesized by B. diazoefficiens are bioactivated in soybean nodules to GA4, modulating nodule size and number in a host-dependent manner [281]. Brassinosteroids, enhanced in alfalfa by E. meliloti NM, were shown to mediate antioxidative responses under PCB stress; inhibition of BR biosynthesis suppressed this protection, confirming BRs as key components of rhizobia-induced detoxification pathways [282]. Together with cytokinins, these hormones integrate with auxin–ethylene signaling to balance root development and nodulation under adverse conditions [283]. Overall, rhizobia act as modulators of host hormonal homeostasis through dual strategies, producing growth-promoting auxins and gibberellins while simultaneously reducing inhibitory ethylene signals. This multifaceted hormonal buffering enables plants to reprogram growth and defense in response to compound stresses, making rhizobia valuable microbial allies for climate-resilient and contaminant-affected agriculture.

Small RNAs (miRNAs) further regulate nodulation through long-distance signaling within the plant. Under drought and salinity stress, miR167 and miR390 are upregulated and suppress auxin-responsive factor (ARF) transcripts, thereby reducing nodule initiation as plants prioritize survival over nitrogen fixation. Conversely, miR172 acts as a positive regulator of nodulation but declines sharply under temperature stress, functioning as a molecular switch that suppresses symbiotic development [284]. Stress also intensifies the autoregulation of nodulation (AON) pathway, where root-derived CLE peptides such as CLE12 and CLE13 move to the shoot and are recognized by receptor kinases including SUNN in Medicago and HAR1 in Lotus [285] (Figure 5). This activates descending inhibitory signals, likely involving cytokinins or miRNAs that suppress further nodule formation and promote early abortion of developing nodules under unfavorable conditions [286].

In addition to hormonal regulation, rhizobial volatile compounds (RVCs) have emerged as important mediators of plant–microbe communication. These include volatile organic and inorganic compounds such as hydrocarbons, ketones, alcohols, ammonia, nitric oxide, hydrogen sulfide, and hydrogen cyanide [287]. Plants perceive these volatile blends and respond by altered gene expression, enhanced root branching, root hair development, iron uptake, and activation of defense pathways associated with induced systemic resistance (ISR) [288]. By modulating root architecture, nutrient acquisition, and stress responses, rhizobial volatiles facilitate colonization and strengthen plant resilience under environmental stress.

Overall, rhizobial symbiosis is regulated by an integrated signaling network involving phytohormones, miRNAs, volatile compounds, and systemic nodulation pathways. Through coordinated hormonal modulation, metabolic signaling, and stress-buffering mechanisms, rhizobia help plants maintain nodulation, growth, and physiological stability under challenging environmental conditions, underscoring their importance for climate-resilient, stress-adapted agriculture.

Figure 5. Schematic overview of long-distance signaling regulating root nodule symbiosis under abiotic conditions. Abiotic stress modulates CLE peptide-, light-, and nitrogen-responsive signaling pathways that coordinate systemic regulation of nodulation through receptor-mediated signaling, phosphorylation cascades, miRNA activity, and ethylene responses. The regulatory modules shown are derived from Lotus japonicas (Lj), Glycine max (Gm), and Medicago truncatula (Mt), while the plant illustration is a representative legume and is not intended to depict any single species. CRISPR-Cas9-mediated editing of cis-regulatory elements is shown as a strategy to modulate promoter activity and improve nodulation efficiency [289].

Figure 5. Schematic overview of long-distance signaling regulating root nodule symbiosis under abiotic conditions. Abiotic stress modulates CLE peptide-, light-, and nitrogen-responsive signaling pathways that coordinate systemic regulation of nodulation through receptor-mediated signaling, phosphorylation cascades, miRNA activity, and ethylene responses. The regulatory modules shown are derived from Lotus japonicas (Lj), Glycine max (Gm), and Medicago truncatula (Mt), while the plant illustration is a representative legume and is not intended to depict any single species. CRISPR-Cas9-mediated editing of cis-regulatory elements is shown as a strategy to modulate promoter activity and improve nodulation efficiency [289].

6.2. Regulation of Oxygen and Nitrogenase Protection

Biological nitrogen fixation in legume nodules is controlled by the “oxygen paradox,” wherein nitrogenase requires strict protection from oxygen despite the simultaneous requirement of oxygen for respiratory ATP production necessary for nitrogen fixation [290]. Nitrogenase is highly oxygen-sensitive because its iron-sulfur clusters are irreversibly inactivated by molecular oxygen, yet nitrogen fixation is an energetically demanding process that depends on intense bacteroid respiration for ATP generation and reducing power [291]. Therefore, nodules maintain a highly regulated microaerobic environment that supports respiration while preventing oxidative damage to nitrogenase (Figure 3).

A major structural adaptation underlying this regulation is the oxygen diffusion barrier (ODB) located within the nodule cortex, which restricts oxygen entry into the infected zone [292]. Oxygen concentrations within nitrogen-fixing tissues are maintained at extremely low nanomolar concentrations, as demonstrated by direct oxygen microelectrode measurements and leghemoglobin-based spectrophotometric analyses of intact nodules [293,294,295]. These kinetic properties enable cbb₃ oxidase to function efficiently within the nanomolar oxygen environment of mature nodules and are now recognized as a hallmark of rhizobial respiratory adaptation during symbiosis [296]. The cortex is differentiated into inner, mid-, and outer cortical regions that, together, regulate oxygen diffusion via controlled intercellular pathways [292]. Structural modifications, including densely packed cortical cells, reduced intercellular air spaces, and glycoprotein-rich occlusions, further stabilize internal oxygen gradients and limit oxygen influx [297]. Under environmental stress conditions such as drought, nitrate exposure, or carbon limitation, rapid osmocontractile responses in cortical cells reduce intercellular spaces and dynamically increase diffusion resistance [298].

Despite these low oxygen concentrations, bacteroids sustain active respiration through specialized high-affinity terminal oxidases. Biochemical characterization of the FixNOQP-encoded cbb₃-type cytochrome c oxidase from Bradyrhizobium japonicum demonstrated an exceptionally high oxygen affinity (Km ~ 4–7 nM O₂), similar to oxygen concentrations measured within infected nodule cells and thereby permitting efficient respiration under microoxic conditions [299]. Mutational analyses further showed that disruption of fixNOQP severely impaired symbiotic respiration, nitrogenase activity, and nodule effectiveness [300,301]. Oxygen-responsive regulatory systems such as FixLJ–FixK and NifA further coordinate transcriptional adaptation to declining oxygen concentrations during bacteroid differentiation and symbiosis establishment [302].

Leghemoglobin (Lb), a plant-derived oxygen-binding hemoprotein present at millimolar concentrations within infected cells, regulates oxygen availability [303]. Leghemoglobin facilitates oxygen transport toward bacteroids and mitochondria while maintaining extremely low free oxygen concentrations compatible with nitrogenase function. Silencing of leghemoglobin genes in L. japonicus caused impaired nitrogen fixation and reduced ATP/ADP ratios despite only minor increases in free oxygen levels, indicating that leghemoglobin functions primarily as an oxygen transporter rather than simply an oxygen scavenger [52].

Maintenance of microoxic conditions inevitably promotes the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) within nodules. Autooxidation of oxy-leghemoglobin generates superoxide radicals and hydrogen peroxide, while mitochondrial respiration and bacteroid electron transport systems contribute additional ROS under hypoxic conditions [304]. To counter oxidative damage, nodules possess robust antioxidant defense systems comprising superoxide dismutase, catalase, glutathione-dependent enzymes, and the ascorbate-glutathione cycle, which in combination maintain cellular redox homeostasis [305]. Nitric oxide generated during hypoxia is detoxified by phytoglobins and bacterial hemoglobins, thereby preserving respiratory efficiency and symbiotic stability [306]. Hydrogen peroxide may also function as a signaling molecule regulating cortical barrier responses and stress-induced structural modifications within nodules [292]. These structural and biochemical mechanisms integrate oxygen diffusion control, respiratory adaptation, oxygen buffering, and antioxidant protection. In stress conditions, this resolves the oxygen paradox and preserves efficient symbiotic nitrogen fixation.

6.3. NODULE INCEPTION (NIN) and NIN-like Proteins: Integrating Nitrogen Signaling with Stress-Responsive Nodulation

The primary member of the NIN-like protein (NLP) family, NODULE INCEPTION (NIN) is a legume-specific RWP-RK transcription factor that functions as a master regulator of root nodule symbiosis [307]. NIN has been functionally specialized to control rhizobial infection, nodule organogenesis, and autoregulation of nodulation (AON) in nitrogen-fixing species, despite the fact that NLPs are evolutionarily conserved in all vascular plants and mainly function in nitrate sensing and the primary nitrate response [308,309] (Figure 6). The common symbiosis signaling pathway initiates infection thread formation, cortical cell division, and the transcription of numerous nodulation-associated genes by activating NIN downstream of the calcium-responsive transcription factor CYCLOPS upon recognition of rhizobial Nod factors by LysM receptor kinases [101,310,311,312,313]. Additionally, NIN directly stimulates rhizobia-responsive CLE peptide genes that mediate long-distance AON communication, balancing the nutritional requirements of plants with the energy demands of symbiotic nitrogen fixation [314,315,316].

The majority of NLPs, in contrast to NIN, are constitutively expressed and serve as key nitrate-responsive transcription factors that combine plant developmental responses with external nitrogen availability [317,318,319]. There is growing evidence that NIN and NLPs have significant functional interaction during nodulation under changing environmental stress conditions. In Medicago truncatula, NLP1 interacts with NIN to control CRE1 expression, thereby connecting nitrate signaling with cytokinin-mediated suppression of nodulation under high-nitrogen conditions [320,321]. In Lotus japonicus, the NLP transcription factor NRSYM1 directly activates CLE-RS2 in response to nitrate. The ability of NLPs to coordinate nitrogen metabolism, transcriptional reprogramming, and stress adaptation has made them significant regulators of drought and cold stress responses in addition to nutrient sensing. This suggests that the NIN–NLP regulatory module integrates environmental and nutritional cues to maximize symbiotic performance [322,323,324,325]. Therefore, improving nodulation efficiency, nitrogen-use efficiency, and legume resilience under climate-induced abiotic stresses can be achieved through modifying NIN/NLP-centered regulatory networks.

6.4. Nitric Oxide and Reactive Nitrogen Species in the Regulation of Stress-Responsive Nodulation

Nitric oxide (NO) is an essential signaling molecule that regulates multiple stages of legume–rhizobium symbiosis, including infection thread progression, nodule organogenesis, bacteroid differentiation, and nitrogen fixation [326]. Transient NO buildup serves as a developmental signal in the early phases of symbiosis, coordinating host cellular responses and rhizobial infection [327]. Through the FixL/FixJ two-component regulatory system, NO affects the expression of over 100 genes in Sinorhizobium meliloti, especially under microaerobic conditions within nodules, according to transcriptomic studies [328]. Genes involved in nitrogen metabolism, such as the nir and nor clusters encoding nitrite and nitric oxide reductases, are further activated by the transcription factor NnrR, which belongs to the Crp/FNR family [329]. However, reactive nitrogen species (RNS), such as S-nitrosoglutathione (GSNO) and per-oxynitrite (ONOO⁻), are produced in excess during prolonged abiotic conditions, such as drought, salinity, flooding, and nutrient constraint, impair NO homeostasis. Increased RNS levels cause tyrosine nitration and protein S-nitrosylation, which alters the function of regulatory proteins and enzymes involved in symbiotic nitrogen fixation and accelerates nodule senescence [330,331,332,333].

Therefore, maintaining NO homeostasis is essential to keeping nodules functional under stress. Class 1 phytoglobins (Pgbs) are one of the major detoxifying mechanisms which work as high-affinity NO scavengers, converting NO to nitrate via the phytoglobin–NO cycle. This prevents excessive nitrosative stress while maintaining the signaling functions of NO [334]. Further, S-nitrosoglutathione reductase (GSNOR) maintains the reversibility and strict regulation of NO-dependent signaling during symbiosis by controlling the amount of S-nitrosylation protein and cellular GSNO turnover [335]. Recent studies further showed that, despite decreased nitrogenase activity, drought-induced hypoxia in Medicago truncatula nodules significantly upregulates Pgb1.1 NR1 and NR2, increasing phytoglobin-mediated NO cycling and ATP generation. According to [336], this metabolic modification reduces nitrosative stress, delays early nodule senescence, and helps maintain nodule functionality in water-limited environments. Moreover, nitrate reductases generated from both plants and bacteria contribute to the synthesis of NO within nodules, demonstrating the symbiotic partners’ coordinated control of NO metabolism [337].

Reversible protein S-nitrosylation has become a significant post-translational mechanism that regulates proteins linked to nitrogen metabolism, symbiotic signaling, and stress adaptation to its detoxifying function. Therefore, increasing nodulation efficiency, extending nodule viability, and maintaining biological nitrogen fixation under climate-induced abiotic stressors can be achieved by designing phytoglobin-mediated NO scavenging or modifying GSNOR activity.

7. Translational Frontiers: Engineering Resilient Symbiosis

7.1. Pan-Genomic Identification of Stress-Adaptive Traits

Advancements in multi-omics technologies have revolutionized the dissection of rhizobial genomes, facilitating the identification of stress-adaptive gene clusters embedded within the expansive rhizobial pan-genome. These efforts underscore the functional plasticity of rhizobia, not merely as nitrogen-fixing symbionts, but as genetically dynamic entities that mediate plant adaptation under environmental constraints. High-resolution genomic, transcriptomic, and metabolomic analyses have uncovered conserved and accessory loci involved in nitrogen fixation (nif), phosphonate metabolism (phn), osmolyte biosynthesis, metal detoxification, and exopolysaccharide production-traits critical for symbiotic performance in stress-prone environments [176,184].

Transcriptomic studies demonstrate that abiotic stressors, including pH fluctuations, osmotic pressure, and metal toxicity, induce global transcriptional reprogramming in rhizobia. Genes encoding pH-responsive transcriptional regulators, membrane transporters, and protective enzymes are differentially expressed, often localized within genomic islands, illustrating the accessory genome’s role in adaptive evolution [338]. Concurrent metabolomic profiling has revealed the accumulation of key osmoprotectants such as trehalose and cadaverine, which contribute to membrane stabilization and osmoadaptation under salinity and drought conditions [339].

Transcriptomic and comparative genomic analyses of sulfate-reducing bacteria, including Desulfovibrio spp., have identified coordinated stress-responsive gene clusters and regulatory modules associated with adaptation to environmental stress conditions [340]. These clusters often comprise transcriptional regulators, sigma factors, chaperones, and detoxification enzymes [341]. and frequently interface with central metabolic and signaling pathways [342]. In rhizobia, such stress-specific modules, including nif and phn clusters, play central roles in ensuring both microbial survival and host fitness. Metabolomic interactions and phytohormonal cross-talk in the rhizosphere are tightly coupled with microbiome dynamics [343]. These interactions orchestrate the recruitment of beneficial microbes and the activation of symbiotic functions in plants.

Mining the rhizobial pan-genome using integrative omics offers a strategic pathway to uncover and engineer core and accessory gene clusters essential for environmental fitness and stress-resilient symbiosis. While these insights support the rational design of synthetic rhizobial inoculants, future research must dissect the functions of individual gene clusters under complex, multifactorial field conditions to enable precision agriculture and resilient soil–plant-microbe systems.

7.2. Synthetic Consortia and Nanomaterial-Assisted Enhancement

Addressing agricultural sustainability under environmental stress increasingly relies on developing stress-resilient microbial consortia adapted to specific agroecosystems. Beyond their established role in biological nitrogen fixation, rhizobia are now recognized as important components of synthetic microbial communities because of their compatibility with diverse rhizosphere microorganisms, adaptability to stressed soils, and participation in horizontal gene transfer [13]. In addition to nitrogen fixation, rhizobia contribute to salinity tolerance, osmotic adjustment, heavy metal detoxification, and oxidative stress mitigation, supporting their use in multifunctional bioinoculant systems [344].

Rhizobia frequently interact synergistically with plant growth-promoting rhizobacteria (PGPR), including Bacillus subtilis, Pseudomonas putida, and Oceanobacillus pseudogrignonense. These microbial consortia collectively express traits such as ACC deaminase activity, siderophore production, phosphate solubilization, indole-3-acetic acid synthesis, and pathogen suppression, which enhance plant growth, nodulation, nutrient acquisition, and stress tolerance [345]. Indigenous rhizobial strains adapted to local soil and climatic conditions often outperform commercial inoculants, emphasizing their importance as reservoirs of stress-adaptive traits for region-specific consortium development [346].

Under saline and nutrient-deficient conditions, co-inoculation of rhizobia with halotolerant PGPR such as P. putida, Bacillus, and Klebsiella improves biomass accumulation, nodulation, and nutrient uptake [347,348]. Certain rhizobial strains additionally alleviate heavy metal toxicity through biosorption, chelator secretion, redox regulation, and antioxidant activation [169]. Exopolysaccharide-producing rhizobia enhance drought and ionic stress tolerance through protective biofilm formation, whereas microbial metabolites, including proline, phenolics, salicylic acid, and antioxidant enzymes, strengthen plant defense responses against oxidative stress [349].

The genomic adaptability of rhizobia, facilitated by mobile genetic elements, supports rapid adaptation to environmental stress, while metagenomic approaches enable monitoring of inoculant persistence and functionality in soils [176,350]. Integration with nanotechnology has further expanded strategies to improve rhizobial colonization, stress resistance, and pollutant degradation (Table 5) [351]. Nano-enabled formulations enhance rhizobial survival through controlled delivery and protection of inoculants, while simultaneously improving plant antioxidant defense, nutrient acquisition, osmotic adjustment, photosynthetic efficiency, and phytohormonal balance, thereby promoting nodulation and biological nitrogen fixation under drought, salinity, and heavy metal stress [352,353,354,355]. Nanomaterials such as nanosilicon and carbon nanotubes stimulate flavonoid and isoflavonoid release, activate rhizobial nod genes, and enhance infection thread formation, nodulation, and nodule biomass [355]. Iron oxide and zinc oxide nanoparticles supply cofactors required for nitrogenase activity, whereas titanium dioxide nanoparticles improve photosynthesis, carbohydrate allocation, and antioxidant enzyme activities, including superoxide dismutase and catalase, thereby protecting nitrogenase from oxidative damage [356].

However, their agronomic performance is strongly influenced by nanoparticle composition, size, surface chemistry, concentration, soil properties, and environmental conditions. Beneficial effects are typically observed within an optimal concentration range, whereas excessive nanoparticle doses may induce phytotoxicity through oxidative stress, membrane damage, impaired root development, disruption of photosynthesis, and metabolic dysfunction, demonstrating a hormetic response in which low concentrations stimulate plant growth and symbiotic performance while higher concentrations inhibit nodulation and nitrogen fixation [357,358]. Moreover, nanoparticle behaviour under field conditions differs substantially from that observed in controlled environments owing to aggregation, dissolution, and interactions with soil constituents, which influence their bioavailability, persistence, and biological activity [359,360]. Increasing evidence also indicates that engineered nanomaterials can modify the abundance, diversity, and functional activity of rhizosphere microbial communities, including beneficial rhizobia and plant growth-promoting microorganisms, with effects varying according to nanoparticle type, concentration, exposure duration, and soil physicochemical characteristics [358,361,362]. Advances in synthetic biology, including CRISPR-Cas systems and modular transcriptional circuits, now enable targeted engineering of stress-responsive pathways [4,363,364,365,366]. Manipulation of genes associated with trehalose biosynthesis, osmoregulation, metal efflux, and EPS production has generated rhizobial strains with improved tolerance to drought, salinity, and heavy metals [367]. Although synthetic biology and CRISPR-based engineering offer promising opportunities to develop stress-resilient rhizobial inoculants, their deployment is constrained by biosafety concerns, as well as regulatory, ethical, and environmental challenges. The environmental release of genetically modified microorganisms requires rigorous assessment of genetic stability, persistence, horizontal gene transfer, and potential effects on native microbial communities [368]. Furthermore, regulatory frameworks differ substantially across jurisdictions, creating uncertainty for the commercialization and adoption of engineered microbial inoculants [369]. Public acceptance, transparent governance, and long-term ecological monitoring are also critical to ensure that the benefits of programmable rhizobia are realized without compromising environmental safety or ecosystem integrity [368,369,370]. Consequently, future development of engineered rhizobia should integrate robust biocontainment strategies, long-term ecological monitoring, and multi-location field validation to address biosafety concerns and ensure that enhanced symbiotic performance is achieved without compromising ecosystem integrity or public confidence.

Table 5. Effects of nanomaterials on Rhizobial Nodulation and Symbiotic Stress Tolerance.

Nanomaterial	Rhizobial Interaction	Stress Type	Mechanism of Action	Outcome	Reference
Zinc Oxide Nanoparticles (ZnO NPs)	Enhances rhizobial nodulation and nitrogen fixation	Salinity Stress	Modulates antioxidant enzyme activities, reduces reactive oxygen species (ROS) generation	Improved growth and stress tolerance in legumes	[371]
FITC-tagged Nano-FeO (nFeO)	Rhizobium with alfalfa (Medicago sativa L.)	Salt stress	Improved Na+/K+ homeostasis, enhanced antioxidant activity, and reduced ROS damage	Enhanced salt tolerance, improved morpho-physiological traits and photosynthetic performance in alfalfa	[372]
Chitosan-GSNO Nanoparticles	Facilitates rhizobial attachment and survival	Drought Stress	Induces systemic resistance, enhances antioxidant defense, promotes biofilm formation	Reduced pathogen load, enhanced symbiosis	[373]
Silver Nanoparticles (AgNPs)	Stimulates rhizobial activity at low concentrations	Heavy Metal Stress	Chelates toxic metals, modulates microbial metabolism	Enhanced legume growth in contaminated soils	[374]
Silicon Nanoparticles (SiNPs)	Improves nodulation efficiency	General Abiotic Stresses	Strengthens cell walls, modulates stress-responsive genes	Increased plant vigor and stress resilience	[375]
Iron Oxide Nanoparticles (Fe3O4 NPs)	Boosts rhizobial iron acquisition	Iron-Deficiency Stress	Enhanced nodulation and nitrogen fixation via improved siderophore mediated Fe acquisition and antioxidant buffering.	Enhanced chlorophyll content and nitrogen fixation	[376]
Manganese ferrite nanoparticles (MF-NPs)	Bradyrhizobium diazoefficiens and Glycine max L. Merr.	ROS	Regulating the nodulation pathway and increasing the number of nodules in soybean.	Nodule number increased without affecting vegetative growth or activating AON pathway	[377]

Table 5. Effects of nanomaterials on Rhizobial Nodulation and Symbiotic Stress Tolerance.

Nanomaterial	Rhizobial Interaction	Stress Type	Mechanism of Action	Outcome	Reference
Zinc Oxide Nanoparticles (ZnO NPs)	Enhances rhizobial nodulation and nitrogen fixation	Salinity Stress	Modulates antioxidant enzyme activities, reduces reactive oxygen species (ROS) generation	Improved growth and stress tolerance in legumes	[371]
FITC-tagged Nano-FeO (nFeO)	Rhizobium with alfalfa (Medicago sativa L.)	Salt stress	Improved Na+/K+ homeostasis, enhanced antioxidant activity, and reduced ROS damage	Enhanced salt tolerance, improved morpho-physiological traits and photosynthetic performance in alfalfa	[372]
Chitosan-GSNO Nanoparticles	Facilitates rhizobial attachment and survival	Drought Stress	Induces systemic resistance, enhances antioxidant defense, promotes biofilm formation	Reduced pathogen load, enhanced symbiosis	[373]
Silver Nanoparticles (AgNPs)	Stimulates rhizobial activity at low concentrations	Heavy Metal Stress	Chelates toxic metals, modulates microbial metabolism	Enhanced legume growth in contaminated soils	[374]
Silicon Nanoparticles (SiNPs)	Improves nodulation efficiency	General Abiotic Stresses	Strengthens cell walls, modulates stress-responsive genes	Increased plant vigor and stress resilience	[375]
Iron Oxide Nanoparticles (Fe3O4 NPs)	Boosts rhizobial iron acquisition	Iron-Deficiency Stress	Enhanced nodulation and nitrogen fixation via improved siderophore mediated Fe acquisition and antioxidant buffering.	Enhanced chlorophyll content and nitrogen fixation	[376]
Manganese ferrite nanoparticles (MF-NPs)	Bradyrhizobium diazoefficiens and Glycine max L. Merr.	ROS	Regulating the nodulation pathway and increasing the number of nodules in soybean.	Nodule number increased without affecting vegetative growth or activating AON pathway	[377]

8. Conclusions

Legume–rhizobium symbiosis is a dynamic and highly coordinated biological system whose resilience under abiotic and chemical stresses depends on the integrated responses of both the host plant and its microsymbiont. This review highlights that successful nodulation and biological nitrogen fixation rely on interconnected regulatory networks governing signal perception, oxygen homeostasis, nitrogenase protection, phytohormonal regulation, exopolysaccharide production, and stress-responsive gene expression. Together, these mechanisms enable symbiotic adaptation to adverse environmental conditions while sustaining plant productivity.

An important conclusion emerging from recent studies is that stress tolerance is not determined by rhizobia alone but by the combined contributions of the host genotype, rhizobial strain, and nodule-associated microbiome. However, substantial inter- and intraspecific variation exists among legumes and rhizobia, and many of the regulatory mechanisms discussed have been elucidated using a limited number of model species under controlled experimental conditions. Therefore, caution should be exercised when applying these findings to diverse legume–rhizobium associations or field environments. A deeper understanding of the conserved and species-specific mechanisms underlying stress-responsive symbiosis will be essential for improving biological nitrogen fixation and developing resilient legume production systems for sustainable agriculture.

9. Future Directions

Future efforts to improve the resilience of legume–rhizobium symbiosis should focus on translating mechanistic insights into field-applicable strategies. Long-term, multi-location field trials are required to evaluate stress-tolerant rhizobial strains and synthetic microbial consortia across diverse soil types, climatic conditions, and host genotypes, as laboratory performance often differs from field outcomes. Standardized evaluation criteria based on nodulation efficiency, nitrogenase activity, stress-responsive gene expression, and rhizosphere persistence will facilitate the comparison and deployment of elite inoculants [378,379].

Advances in multi-omics, synthetic biology, and CRISPR-Cas genome editing provide unprecedented opportunities to engineer stress-responsive pathways involved in osmoregulation, exopolysaccharide biosynthesis, oxygen regulation, and nitrogen fixation, thereby improving rhizobial persistence and symbiotic efficiency under adverse conditions [4,366]. Systems biology approaches, including metabolic flux analysis and genome-scale modelling, will further support the rational design of strains with enhanced stress tolerance and metabolic performance [380]. Future bioinoculants should also integrate beneficial non-rhizobial endophytes and arbuscular mycorrhizal fungi [5,203] to develop functionally stable microbial consortia capable of sustaining biological nitrogen fixation under environmental stress [381].

Finally, integrating improved inoculant formulations with precision agriculture, artificial intelligence-assisted prediction models, and environmentally compatible delivery systems, such as seed coatings and controlled-release formulations, will enhance inoculant establishment and field performance [382,383,384]. Coupled with appropriate biosafety frameworks and ecological risk assessment [385,386], these multidisciplinary approaches will accelerate the development of climate-resilient bioinoculants for sustainable agriculture.