Biological nitrogen fixation gives plants a competitive edge by converting atmospheric nitrogen into ammonium. Microorganisms that produce the nitrogenase enzyme can carry out this activity (
Vessey, 2003). Rhizobium is a crucial element in legumes, as it forms root nodules that facilitate atmospheric nitrogen fixation, which is beneficial for the plant
(Arya et al., 2024). Rhizobia and leguminous plants have symbiotic relationships in which the latter produce nitrogen-fixing nodules in their roots. Symbiotic interactions between compatible legume host plants and rhizobia involve precise molecular communication between the two partners
(Dudeja et al., 2025). Only a limited number of plant species exhibit this special relationship (
Spaink, 2000). Rhizobial bacteria infiltrate leguminous plants through signal exchange, enabling the bacteria to enter the roots and develop into ammonia-converting forms, the nodules (
Jones et al., 2007). When rhizobium bacteria and legume crops coexist, the bacteria can infect plant roots and form root nodules, which provide nutrients for crops (
Samudin and Kuswantoro, 2017). Various results revealed that plant height (cm), number of effective tillers per m
2 and leaf area index (LAI) are significantly influenced by the combination of nitrogen levels and microbial inoculation treatments
(Kumari et al., 2025).
According to some estimates, biological nitrogen fixation is responsible for the fixation of up to 200 metric tons of nitrogen annually in the agricultural system, representing a major saving in the cost of crop production both financially and as well as environmentally. It converts atmospheric nitrogen into ammonium, a form that is readily utilized by plants. The ability of some plant species to supply some, if not all, of their requirement for nitrogen in this way gives them a substantial competitive advantage over those that lack this ability. Microorganisms that produce the enzyme nitrogenase can carry out the process of nitrogen fixation. In a very restricted group of plant species, the association between the host plant and the bacterial symbiont is a highly intimate one and the bacteria are housed within nodules, which is a specialized organ that forms in the root (
Vessey, 2003).
Under low nitrogen environments, the host roots release particular flavonoids that cause rhizobia to produce particular lipo-chito-oligosaccharide signalling molecules known as Nod factors. The rhizobia perceive flavonoids through NodD, a protein that stimulates the transcription of bacterial Nod genes involved in the production and secretion of Nod factor
(Spaink et al., 1989; Mulligan and Long, 1985). Many legumes produce specific flavonoids that only induce Nod factor production in homologous rhizobia and therefore act as important determinants of host range (
Liu and Murray, 2016). A type of flavonoid called isoflavones is primarily found in leguminous plants. It is essential for interactions between plants and microbes, such as symbiosis between rhizobia and legumes and defensive mechanisms
(Mazur et al., 1998; Sugiyama, 2019). According to
Subramanian et al., 2006, isoflavones stimulate the nodulation genes in leguminous plants, which helps in the nodulation process.
This review has been done to compile the various factors responsible for BNF (Biological nitrogen fixation) and highlights the role played by flavonoids in the BNF process of leguminous plants. Flavonoids are the key signaling molecules that initiate and regulate the symbiosis between rhizobia and legumes.
This review article is based on literature search and data analysis. Abstracts and full research papers were systematically reviewed; databases like Web of Science, Google Scholar and PubMed were thoroughly searched for research papers and related articles published till 2025.
Legume-rhizobium symbiosis
The effective symbiosis between rhizobia and legumes depends on the Nod factors, flavonoids, bacterial surface polysaccharides and plant lectins
(Pindi et al., 2020). Nod factors allow the rhizobia to enter the host plants and stimulate flavonoids from legume roots. Once bacteria enter the legume roots, extracellular polysaccharides and proteins are exchanged between symbionts and amplifying the response to inoculation (
Broughton, 2003). The specific exudation of flavonoid mixtures from legume hosts and NodD proteins from different rhizobia is partially responsible for the host specificity of symbiosis
(Gibson et al., 2008; Skorupska et al., 2010; Rose et al., 2012; Sharma et al., 1993).
Flavonoid metabolism in leguminous plants
Flavonoids are a large (≈9000) structurally diverse class of phenolic compounds found in all higher plants and classified into one of the following groups: flavones, isoflavonoids, flavonols, flavandiols, proanthocyanidins and anthocyanins (
Ferrer et al., 2007;
Kumar et al., 2024). Flavonoids are crucial signalling molecules in the symbiosis between legumes and their nitrogen-fixing symbionts, the rhizobia (
Liu and Murray, 2016). Flavonoids, secreted by legume roots, act as chemical messengers, regulating physiological processes and inhibiting cell cycle
(Kumari et al., 2025). Flavonoids in symbiosis attract rhizobia, induce Nod gene synthesis, determine host specificity and regulate root development. Isoflavonoids attract rhizobia to legume root nodules, establishing symbiotic relationships that enhance plant growth (
Abd-Alla et al., 2023). There are seven different subclasses of flavonoids based on their structural differences, which are presented in Table 1.
Flavonoids act as chemoattractants
Flavonoids act as chemical signals to attract the rhizobium bacteria. They are frequently exuded into the rhizosphere to attract compatible rhizobia that reside in the rhizosphere, promote or suppress the expression of rhizobial nod genes, suppress root pathogens, promote the germination of mycorrhizal spores and hyphal branching, influence quorum sensing and chelate soil nutrients
(Broughton et al., 2003; Cooper, 2004;
Martens and Mithofer, 2005). The rhizobial membrane absorbs flavonoids, in the form of aglycone, after they are released, potentially
via porins
(Kobayashi et al., 2004; Wang et al., 2012; Taylor and Grotewold, 2005).
Flavonoids act as a selective agent for compatible symbiotic organisms
The symbiosis between
Rhizobium spp. and legumes is highly host-specific. Only a certain strain of
Rhizobium spp. can successfully establish a symbiotic connection with a limited number of host plant species
(Gyorgypal et al., 1991; Hassan and Mathesius, 2012). It has been demonstrated that the NodD protein produced by a
Rhizobium spp., a strain with a wide host range, interacts with more flavonoids than does NodD produced by a strain with a confined host range (
Yokoyama, 2008). This affinity between NodD and flavonoids partially dictates the host range. The combination of flavonoids present in the root exudate of legume species acts as a selective agent for compatible symbiotic organisms (
Maxwell et al., 1989). Recent studies suggest that production of key “infection” flavonoids is highly localized at infection sites and are phytoalexins that may have a role in the selection of compatible symbionts during infection (
Liu and Murray, 2016).
Flavonoids activate the bacterial regulatory protein NodD and nod gene expression
Flavonoids from roots and seeds induce bacterial nodulation genes in legume-rhizobia symbioses, controlling lipo-chitooligosaccharide signals to host plants. However, successful symbiotic development requires the transmission of a variety of other signals from rhizobia to plant roots and flavonoids initiate the production of most of them (
Cooper, 2004). Flavonoids activate the bacterial regulatory protein NodD in nitrogen-fixing symbiosis. This protein functions as a plant morphogen that can initiate nodule formation by controlling the nod regulation required for the synthesis of the bacterial Nod factor (
Dénarié and Roche, 1992;
Schultze et al., 1994; Fisher and Long, 1992). The binding of a flavonoid to NodD protein enhances transcription of nod genes by facilitating RNA polymerase access, forming a nod box with target DNA sequences (
Long, 1996;
Spaink et al., 1989; Mulligan and Long, 1985). NodD recognizes a specific promoter sequence, the Nod box and NodD binds to this promoter element, resulting in transcriptional activation of the downstream genes (
Stacey, 2007). The produced NodD is recognized by plant LysM receptor-like kinases, which causes root tip curl and encloses symbiotic cells and thereby fixing nitrogen.
Nodulation strategies in legume symbionts
The legume family (Fabaceae) is the third largest family of flowering plants, with members of more than 650 genera, 18,000 species spread around the globe (
Frankow-Lindberg and Dahlin, 2013). Their evolutionary success is due to their symbiosis with the
Rhizobium spp., which enables the plants to tolerate nitrogen-deficient soils. In natural ecosystems, the quantity of nitrogen fixed by legumes is estimated to be 28-84 kg per hectare per year, while during a cropping environment, this can rise to several hundred kilograms per hectare (
Catherine and Joel, 2018). Rhizobia follow three nodulation strategies during the process of nodulation: Nod, T3SS and non-Nod/non-T3SS. In the Nod strategy, strain-specific lipochitin oligosaccharides (LCOs) called Nod factors (NFs) are produced under the control of
nod genes. NFs are perceived by LysM-receptor-like kinases that activate the common symbiotic signalling pathway (CSSP). In the second strategy, the T3SS strategy, T3SS effectors activate CSSP components by bypassing NF recognition. The third nodulation strategy is still unknown, but it involves neither
Nod nor T3SS functions and occurs
via CSSP activation (Fig 1). CSSP activation is thus the primary nodulation strategy. Nod-independent and T3SS-independent nodulation (the third strategy) occurs
via CSSP activation, since most CSSP components have been identified in
Aeschynomene evenia, which is only nodulated
via this strategy. It is well established that flavonoids released by the roots of legume species regulate the Nod strategy (
Abdel-Lateif et al., 2012).
A well-studied effect of root-exuded flavonoids is their regulation of
Rhizobium spp.
nod genes (
Cooper, 2007). Luteolin in
Medicago sativa and 7,4’ dihydroxyflavone in
T. repens were found to act as nod genes inducers
(Redmond et al., 1986). Typically, nanomolar to low micromolar range concentration of flavonoids is required for this induction and mixtures of different flavonoids are more effective than a single compound
(Moscatiello et al., 2010). Nod factors required for nodule formation are produced by the cooperative action of several
nod gene products. These
nod genes are regulated by NodD, a LysR family transcription factor. Binding of an appropriate flavonoid to NodD facilitates the access of RNA polymerase and thereby enhances the transcription of the
nod genes (Fig 2). The NodD-flavonoid complex binds to its target DNA sequences, known as a
nod box. The utilization of flavonoids by
Rhizobium spp. is associated with a rise in the concentration of cellular calcium level, which acts to induce the expression of NodD
(Wang et al., 2011). The secreted
nod factors are recognized by the plant’s LysM receptor-like kinases, which triggers the characteristic curling of the root hair tip back on itself, thereby trapping the symbiont cells within a particular area, from which they are taken up into the root proper
via an infection thread
(Horvath et al., 1993). Once the inner root is reached, they are endocytosed into nodule cells and consequently initiate the process of nitrogen fixation. The cell division process is also induced by Nod factor, as well as gene expression in the root cortex and pericycle, which initiates the development of the nodule
(Cullimore et al., 2001; Okazaki et al., 2013).
Flavonoids in root nodule development
Nodule development involves pre-infection, initiation and differentiation stages, with legume roots’ flavonoids activating rhizobial nod genes and host plant flavonoids affecting nodule formation (
Hirsch, 1992). Flavonoids play a significant role in modulating auxin transport during the initiation of nodule primordia in plants. Genetic studies in
Arabidopsis show that flavonoid-null mutants have increased root auxin transport, which can be complemented by external application of flavonols. Flavonoids interact with auxin transporters through Pin-formed gene homologs and
in vitro studies show that they affect polar auxin transport similarly to synthetic chemical inhibitors
(Peer et al., 2004; Taylor and Grotewold, 2005).
The first indirect evidence for an involvement of auxin in nodulation came from experiments showing that external manipulation of auxin transport with synthetic auxin transport inhibitors can result in the formation of nodule-like structures
(Hirsch et al., 1989). Auxin, synthesized in the shoot, is transported to roots
via polar auxin transport, where levels can be regulated through breakdown, conjugation, or transport (
Bandurski et al., 1995;
Lomax et al., 1995). Flavonoids control nodule development and differentiation by inhibiting auxin transport and promoting localized accumulation at the nodule initiation site, potentially influenced by nod factor perception.
Flavonoids may regulate nodule number in symbioses, as evidenced by reduced isoflavonoid formononetin and its glycoside ononin in
Rhizobium spp.-induced and AM-induced symbioses. Flavonoids actively control symbiosis, with higher accumulation of isoflavonoids in wild-type common bean roots. Exogenous supply of daidzein or coumestrol increases nodule number (
Abd-Alla, 2011;
Noorden et al., 2006; Hayashi, 2012).
