Key Message We mapped key regions of the maize genome that influence the formation of aboveground (aerial) roots, which in some varieties have been associated with symbiosis with nitrogen-fixing bacteria. Abstract Modern agriculture relies heavily on chemically synthesized nitrogen fertilizers, which ensure high yields but also carry high economic and environmental costs. Biological nitrogen fixation (BNF) supplies high amounts of nitrogen to legumes, and several avenues of research are underway to extend it to cereal crops. In maize, aerial roots formed in Sierra Mixe landraces have been associated with BNF. However, much of the genetics underlying aerial root morphology remains unknown. Here, we evaluate aerial root morphology traits associated with BNF in three segregating populations derived from crosses between two Midwest-adapted inbred lines and three landraces. Inclusive composite interval mapping (iCIM) with flowering time as a covariate identified 37 quantitative trait loci (QTL) for three aerial root traits (nodes with roots, root size, and roots per node) which exhibit moderately high heritability (H2 = 0.65 to 0.83). The combined proportion of phenotypic variance explained by the detected QTL ranged from 23 to 51%, depending on the trait and population, and potential candidate genes were identified through literature searches, macrosynteny, and gene expression analyses. Introgressing the most relevant aerial root-associated QTL into elite genotypes may provide a path toward achieving meaningful levels of BNF-associated traits in maize, but further work is needed to assess this approach's viability under field conditions.

Nitrate impairs both symbiotic and free-living biological nitrogen fixation (BNF). While nitrate-induced phosphorylation signalling has been implicated in the inhibition of symbiotic BNF, the suppression of free-living BNF has generally been attributed to ammonium generated during nitrate assimilation. However, whether nitrate can inhibit free-living BNF independently of ammonium feedback regulation remains unclear. Here, an ammonium-deregulated mutant of Azotobacter chroococcum (A4) was used to investigate whether nitrate inhibits nitrogen fixation independently of ammonium regulation. Despite the loss of ammonium-mediated inhibition, nitrate significantly suppressed nitrogen fixation. Nitrate at concentrations above 2 mM reduced extracellular ammonium accumulation, with 10 mM nitrate decreasing ammonium production to 61% of that observed under nitrogen-free conditions. Integrated multi-omics analyses revealed that nitrate triggered extensive regulatory reprogramming across multiple molecular layers, with both coordinated and layer-specific responses across transcriptomic, proteomic and phosphoproteomic levels. These responses differed from the typical ammonium-mediated feedback regulation characterized by substantial repression of nitrogen fixation-related genes or proteins, but nevertheless resulted in reduced ammonium excretion, accompanied by enhanced biomass accumulation and extracellular polymeric substance (EPS) production in A4. Together, these findings indicate that, rather than directly repressing the nitrogen fixation machinery, prolonged nitrate exposure suppresses nitrogen fixation output through global regulatory reprogramming that redirects cellular metabolism and resource allocation away from nitrogen fixation. This study provides new insights into nitrate-mediated regulation of free-living diazotrophs and has implications for optimising nitrogen management and improving the application of nitrogen-fixing microorganisms.

Every cell must decide what enters the nucleus and what stays out. The nuclear pore complex (NPC), built from proteins called nucleoporins (NUPs), spans the double membrane of the nuclear envelope and shuttles molecules between the cytoplasm and the nucleus. Most NPC components serve this universal housekeeping role, so it was striking when a handful turned out to matter specifically for symbiosis. The NUP107-160 subcomplex, which forms the outer ring of the pore (Tamura et al. 2010), was first linked to symbiosis in the model legume Lotus japonicus. Mutants in 3 of its members, NUP133, NUP85, and NENA, show impaired root nodulation and arbuscular mycorrhization. These mutants also lack the nuclear calcium spiking triggered by host perception of symbiotic microbes (Kanamori et al. 2006; Saito et al. 2007; Groth et al. 2010). This calcium spiking requires nuclear envelope-localized cation channels, CASTOR and POLLUX, in Lotus (Charpentier et al. 2008). The same pathway operates in the related model legume Medicago truncatula, where the Pollux ortholog DMI1 fills this role (Ané et al. 2004). The function of these channels likely depends on reaching the inner nuclear membrane, but how these NUPs influence symbiotic signaling has remained unclear for nearly 2 decades.

Nitrogenase plays a critical role in biological nitrogen fixation. However, current methods for detecting nitrogenase activity are labor-intensive and unsuitable for large-scale applications. To address this issue, we developed a real-time visual detection system for nitrogen-fixation ability in soybean (Glycine max) nodules using reporter gene-tagged rhizobial strains. Among the four reporters tested, the LUX reporter gene provided the most promising results for deep-tissue imaging because, unlike GFP and RFP, it suffers no autofluorescence interference, and unlike GUS, it does not require exogenous substrate. Further screening identified the nitrogenase-correlative promoter pc16350, which drove LUX expression with high sensitivity at levels strongly correlated to nitrogenase activity. This system enables the rapid, non-invasive monitoring of nitrogen-fixation activity in soybean nodules.

Mycorrhizal fungi form essential symbiotic relationships with plant roots, facilitating nutrient exchange and promoting plant health. Understanding their interactions can benefit from advanced imaging techniques capable of visualizing nutrient exchange and structural colonization at subcellular resolution across large sample sizes. This review explores novel imaging approaches that are revolutionizing our understanding of root–mycorrhizal fungal symbioses. Several techniques can now visualize and characterize mycorrhizal fungi and associated root structures non-destructively and in three dimensions, for example X-ray computed tomography (micro-CT), X-ray fluorescence (XRF), and X-ray absorption near edge structure (XANES) spectroscopy. Metabolic processes and nutrient exchange can be tracked through positron emission tomography (PET), fluorescent nanoparticles (FNPs), and the monitoring of electrical signalling. Artificial intelligence (AI)-powered image processing software is enabling high-throughput analysis of complex images generated from a range of sources. Mycorrhiza systems are also able to be tracked in-field at multiple scales: hyperspectral imaging can detect mycorrhizal associations at the kilometre scale, while portable MRI imagers can detect changes at the tissue scale. These converging technologies enable the direct, continuous measurement of structural and metabolic root–mycorrhizal fungi interactions, paving the way for a mechanistic understanding of these vital symbiotic partnerships and their impact on plant health and ecosystem functioning.

Root nodule organogenesis requires the activation of symbiotic developmental programs within a spatially restricted region of the root. Although many regulators of nodule initiation have been identified, the mechanisms that delimit the domain of organogenic cell proliferation remain poorly understood. In this study, we show that the AP2/ERF transcription factors PUCHI1 and PUCHI2 function in spatially restricting cortical cell division during nodulation in Lotus japonicus. The loss of PUCHI function increased infection thread formation and nodule primordium initiation; however, it did not increase the number of mature nodules. Instead, puchi1 and puchi2 mutants frequently formed clustered nodules accompanied by ectopic cortical cell divisions surrounding developing primordia. Constitutive activation of CCaMK induced broadened spontaneous proliferative structures in the mutant background even in the absence of rhizobia, indicating that this phenotype was not simply a consequence of enhanced infection. PUCHI1 expression was induced during early symbiotic signalling downstream of the NODULE INCEPTION (NIN)–associated transcriptional network; both PUCHI genes were preferentially expressed in the basal region of developing primordia, corresponding to sites of ectopic proliferation in the mutant. Transcriptome and reporter analyses suggested that PUCHI regulated auxin-response patterning, potentially via STY1-, YUCCA11-, TAR2-, and Forked1-like-associated pathways. Additionally, PUCHI1 promoted NIN expression via the cytokinin-responsive CE region of the distal promoter independently of LHK1-mediated cytokinin perception. These findings indicate that PUCHI genes define the spatial domain of organogenic cell proliferation during nodulation; they coordinate auxin-related patterning with the NIN transcriptional module to ensure localised nodule formation.

Root arbuscular mycorrhizal symbiosis (AMS) allows plants to thrive in nutrient-deficient environments. This symbiosis shaped land life evolution and allowed the emergence of root nodule symbiosis (RNS). Both AMS and RNS involve internalization of symbionts—mycorrhizal fungi and nitrogen-fixing bacteria, respectively—in either root ground tissue or new derived cells. This internalization relies on root cellular reprogramming, which includes ectopic cell cycle activation in AMS, or extensive cell cycle activation in RNS. Cell divisions and meristematic activity in the roots are accompanied by ribosome biogenesis and active mRNA translation. Whereas arbuscular-infected cells show enrichment in ribosomal proteins (RPs), transcripts encoding RPs accumulate at early stages of RNS. Specific components of the translational machinery, including eukaryotic initiation and elongation factors, are detected in single-cell transcriptomes of actively dividing cortical cells that will give rise to a nodule primordium. The diversity of heterogeneous ribosomes and their regulatory associated components might contribute to the translation of specific mRNA subsets in different tissues, explaining the differences between transcriptome- and polysome-associated mRNAs observed in early RNS. Features of the regulated mRNAs such as upstream ORFs may have an impact on translation initiation. Furthermore, evidence suggests that translation is modulated by small and long non-coding RNAs. We discuss the relevance of translation in association with cellular reprogramming in root symbioses.

Nitrogen is often a limiting nutrient for agricultural crops. Most microbes also require an external fixed-nitrogen source for optimal growth. Even for microbes capable of biological nitrogen fixation (BNF), it is generally the case that they will regulate their metabolism to prioritize assimilation of fixed-nitrogen. Microbes employ various strategies and pathways to take advantage of available fixed-nitrogen compounds found in their natural environments. Azotobacter vinelandii is a model microbe for the study of BNF. Due to the high energetic cost of the process, BNF in A. vinelandii is repressed in the presence of ammonium, urea and nitrate. Prior studies indicated that strong inhibition of nitrogen fixation by urea and nitrate in A. vinelandii is actually the result of intracellular conversions of these metabolites into ammonium. In this study, we demonstrate a strategy to eliminate BNF inhibition by both urea and nitrate at concentrations ranging as high as 15 mM in a strain lacking the genes for urease and nitrate reductase, resulting in continued nitrogenase activity in the presence of these common fertilizer inputs. In contrast to the properties of A. vinelandii, the diazotroph Gluconacetobacter diazotrophicus naturally lacks these pathways, prompting the question of whether urea or nitrate inhibit BNF or support growth in G. diazotrophicus. To probe this observation, we developed experiments to demonstrate that while the presence of urea and nitrate delay the initial growth rate in G. diazotrophicus, nitrogenase activity and ammonium accumulation occurs at a similar rate in the presence of these metabolites. These results indicate that biological nitrogen fixation in G. diazotrophicus is somewhat insensitive to these nitrogen sources. This illustrates that alternative pathways in diazotrophic strains should be carefully considered in any efforts to optimize extracellular nitrogen production for biofertilizer applications and strain optimization, and that additional design strategies can be effective to assure that diazotrophs continue to fix nitrogen in the presence of specific nitrogen compounds common to industrial fertilizers.

Beneficial plant–microbe associations (BMAs) offer a valuable opportunity to reduce dependence on synthetic inputs. However, traditional breeding has rarely targeted traits that enhance beneficial interactions, and conventional agricultural practices have often degraded soil health and microbial diversity. We present a framework that combines breeding for traits that facilitate BMA with soil management practices that enrich BMA. This approach integrates advanced breeding technologies with strategies for precise production and inoculation of microbes. Strengthening these complementary plant- and microbe-centered approaches and encouraging their adoption by farmers can foster more resilient and productive agricultural systems with reduced dependence on pesticides and fertilizers.

Plants preferentially absorb phosphorus (P) as orthophosphate/ inorganic Phosphate (Pi), which has low solubility and is readily fixed in soil. This generates the P limitation condition in many soils, for which plants have evolved a number of responses collectively known as the Phosphate Starvation Response (PSR). Significant advancements have been achieved in identifying PSR, and a holistic viewpoint is presented in this review to summarize responses to Pi starvation and their regulation. Plant response to Pi starvation is mediated by a plethora of players, including MYB transcription factors, strigolactone, malate, and brassinosteroids. The involvement of Arbuscular Mycorrhizal Fungi (AMF) in the process adds another dimension for enhancing PSR. Strigolactone secretion from the root increases AMF colonization and nodulation for enhancing Pi uptake. Pi uptake can be achieved directly by the plant or indirectly by the Pi transporter and AMF. Root system architecture changes according to the Pi present in the environment. Increased auxin signalling is associated with Pi starvation, playing a part in lateral root development. Overall, several unanswered questions remain in the phosphate starvation response of plants, especially from the perspective of AMF symbiosis. Research focused on enhancing PSR with symbiotic association with AMF using advanced biotechnological approaches would pave way for increasing crop productivity.

Arbuscular mycorrhiza (AM) represents a key strategy for plants to overcome nutrient starvation. In legumes and rice, the development of this symbiosis requires the GRAS transcription factor DELLA, previously identified as central proteolytic target of gibberellin (GA) signaling. DELLA performs critical functions across multiple stages in AM development, including inner root cortex patterning, arbuscule initiation and degeneration, by regulating essential downstream genes driving these phenomena. Moreover, DELLA appears as a regulatory hub that integrates hormonal signals, environmental stimuli, and symbiosis formation. Here, we highlight recent advances in our understanding of DELLA-mediated regulation of AM development and provide state-of-the-art insights into how DELLA orchestrates these signaling pathways.

Legumes acquire nitrogen via a symbiotic interaction with diazotrophic rhizobia bacteria. In return for getting fixed nitrogen, plants deliver high amount of photosynthate to the bacteria to support the nitrogen fixation process. Hence, biological nitrogen fixation in legume plants is a highly energy-demanding process that relies on the precise coordination of carbon allocation and metabolism between the host plant and its microbial symbiont. Although significant progress has been made in understanding carbon fluxes during nodulation, how these processes are spatially and functionally organized across different cell types and developmental stages within nodules remains poorly resolved. This limitation has hindered a comprehensive understanding of how carbon metabolism supports the establishment, maintenance, and termination of symbiosis. In this review, we explore the current understanding of carbon transport and metabolism throughout the nodulation process, from early allocation during rhizobial infection to the complex metabolic, transport, and regulatory networks in mature nitrogen-fixing and senescing nodules. We highlight key knowledge gaps, especially regarding cell-type-specific and spatial regulation of carbon metabolism. Finally, we discuss how emerging single-cell and spatial omics techniques offer powerful tools to resolve these gaps, enabling a deeper understanding of the metabolic and regulatory complexity that underpins legume–rhizobia symbiosis.

Plants are found in dynamic soil ecosystems, which are highly enriched with microorganisms that have significant impacts on the health and productivity of plants. Promotion of nutrient uptake, fixation of nitrogen, and tolerance to stress by arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria occur via complex molecular communication with plant roots. Post-transcriptional gene silencing is mediated by MicroRNAs (miRNAs)-small non-coding RNAs that control gene networks in symbiosis and stress responses. Concurrently, phytohormones (auxin, cytokinin, ethylene, jasmonates, salicylic acid, abscisic acid, strigolactones, gibberellins, and brassinosteroids) control root development, defence, and microbial recruitment. Their combined regulatory functions in the establishment of root symbiosis are yet to be fully appreciated, although the integrated regulatory functions of miRNAs and phytohormones in regulating root symbiotic assembly have increasingly been recognised. The paper is a synthesis of the existing knowledge on root-associated beneficial symbioses and a critical assessment of the new roles of plant miRNAs and phytohormonal pathways in the establishment and maintenance of nodulation and arbuscular mycorrhizal associations. We suggest that miRNAs and phytohormones are linked in a complex regulatory network in which miRNAs can regulate hormone pathways and vice versa. We have also demonstrated crucial knowledge gaps by identifying molecular crosstalk between plants and their microbiota, functional validation of miRNA-target pairs, cross-kingdom small RNA trafficking and systemic shoot–root signalling that future studies can use to harness the full potential of plant–microbe interaction for achieving sustainable agriculture.

Ethylene modulates plant fitness, but its role in rhizosphere soil multifunctionality remains unclear. This study demonstrates that endogenous ethylene suppresses C- and N-cycling functions while promoting organic P mineralization in the maize rhizosphere, ultimately diminishing the overall soil multifunctionality and attenuating its phenologically driven peak at the tasseling stage. Ethylene reshaped microbial community assembly by enriching opportunistic taxa (e.g., Actinobacteria) while reducing the diversity and relative abundance of sensitive taxa. Community composition (e.g., Bacillus) and α diversity of ethylene-sensitive taxa were negatively correlated with soil multifunctionality. Inoculation with ACC deaminase-producing Bacillus pumilus and Streptomyces gardneri (opportunistic strains) significantly elevated C- and N-cycling enzyme activities and boosted maize growth. However, a direct causal link between ACC deaminase production and these effects requires further experimental validation. Collectively, these findings elucidate that ethylene drives rhizosphere biogeochemical trade-offs through the selective filtering of microbial functional guilds, providing a theoretical foundation for rhizosphere microbiome management in crop production.

Nodule organogenesis is a complex developmental process which is induced post-embryonically following rhizobia infection and relies on coordinated action of a suite of transcription factors at multiple stages starting from its initiation, de novo meristem establishment and differentiation. The rewiring of nitrate-responsive NIN-LIKE PROTEIN (NLP) homolog, NODULE INCEPTION (NIN) transcription factor through genetic adaptations in the common ancestor of the nitrogen-fixing clade is crucial to the origin of nodulation trait. Moreover, nodule formation emerged through the co-option of evolutionary ancient root/lateral root developmental pathways mediated by conserved transcriptional regulators such as NF-Y, LBD16, SHR/SCR, WOX5, PLT and KNOX. Importantly, NIN has acquired functions to integrate signals from rhizobia infection and divert the existing developmental pathways towards nodule organogenesis, thereby acts as a master regulator of root nodule symbiosis. Nodule specific innovations in the existing developmental pathway genes through mechanisms like genetic adaptations in their cis-regulatory region, paralog retention, changes in spatio-temporal gene expression pattern and functional changes to the protein are crucial for imparting developmental novelty during nodule organogenesis. Here, we provide a consolidated idea on transcription factor–mediated genetic modules regulating distinct stages of nodule organogenesis with an emphasis on their nodule specific innovations. This knowledge gain is particularly important to engineer nitrogen-fixing nodules into non-nodulation crop plants which will eventually minimize the reliance on synthetic nitrogen fertilizers and thereby favors a sustainable agricultural system.