Mutualism in the symbiosis between arbuscular mycorrhizal fungi and plants is based upon the exchange of carbon for soil minerals, with phosphate being of central importance. The exchange of nutrients occurs when the fungus transiently colonises root cells, producing hyphal structures called arbuscules. The movement of phosphate from fungus to plant is well established, however its coordination and regulation at the ephemeral arbuscules remains elusive. Here, non-invasive imaging captures the complete growth and collapse of the arbuscules in unprecedented resolution, revealing heterogeneity in arbuscule development. Tracking the dynamics of rice PHosphate Transporter 1;11 (OsPHT1;11/ PT11) as a proxy for symbiotic phosphate transport shows consistent localisation across diverse arbuscules. However, we uncover phosphate-responsive variability in PT11 abundance, representing an essential, cellular-level layer of nutrient regulation. Such plasticity in arbuscule phosphate uptake capacity evidences uncoupling of arbuscule presence and arbuscule function, thereby demonstrating that arbuscules are not identical units of nutrient exchange.

The search for life now extends beyond the traditional habitable zone to include the icy moons of Jupiter and Saturn. These moons feature ice-covered surfaces overlying substantial oceans formed primarily of liquid water and other potential constituents, such as ammonia. On Earth, ammonia supports biochemistry at low concentrations by providing nitrogen but becomes disruptive at higher concentrations. Ammonia could therefore influence the habitability of extraterrestrial oceans, yet this topic has received limited attention in the literature. This review synthesises current research on ammonia in Saturn’s icy moons, Enceladus and Titan, and its effects on terrestrial life. We summarize the celestial incorporation, speciation, and phase behaviour of ammonia and review data on its occurrence and concentration in icy moon oceans. We examine the role of ammonia in prebiotic chemistry, biochemistry, and toxicity. Focusing on bacteria, we compare known survival limits in ammonia to estimated ammonia concentrations on Enceladus and Titan. We find that bacterial survival limits exceed concentrations estimated on Enceladus, but are below those estimated on Titan, and propose that ammonia measurements are crucial for assessing extraterrestrial habitability. Finally, we highlight outstanding knowledge gaps and challenges that influence our understanding of how ammonia shapes the potential for life beyond Earth.

Desert ecosystems in Kuwait are increasingly affected by land degradation, resulting in nutrient-limited soils that constrain native plant establishment. Harnessing indigenous diazotrophic bacteria adapted to arid environments may provide a sustainable strategy to improve plant growth and nutrient acquisition. Free-living and root-associated nitrogen-fixing bacteria contribute substantially to nitrogen inputs in arid ecosystems and may enhance plant growth, performance and nutrient acquisition under nutrient-poor conditions. This study evaluated the growth performance and nutrient uptake ability of four native plant species of Kuwait following inoculation with a consortium of selected indigenous putative diazotrophs isolated from the Kuwait desert soils. The seedlings of Vachellia pachyceras were inoculated with both indigenous root-nodule bacteria isolated from Kuwait desert and a commercial inoculum to evaluate their symbiotic efficiency. The seedlings were cultivated under greenhouse conditions using either native desert soils or a potting mix substrate to assess the influence of growth medium or inoculation response. Across species, inoculation significantly enhanced plant dry mass and nutrient uptake compared to the non-inoculated controls. The magnitude of improvement varied among bacterial density, host plants, and growth substrate. These findings support the potential use of indigenous diazotrophs as biofertilizers to enhance plant growth and nutrient uptake of native plant species, and for restoration and revegetation efforts in arid environments. However, direct measurements of nitrogen fixation were not conducted and should be addressed in future field-based studies. This study represents the first evaluation of Kuwait’s native seedlings inoculated with indigenous diazotrophs, highlighting their potential for sustainable ecosystem restoration.

Reprogramming of differentiated root cortical cells into proliferative stem cells is a prerequisite for legume nodule organogenesis, yet the molecular trigger that confers stem-cell identity upon these cortical cells remains elusive. Here we demonstrate that, in soybean (Glycine max), the canonical root stem-cell regulator WUSCHEL-RELATED HOMEOBOX gene WOX5 is activated by rhizobia specifically in cortical cells that will give rise to nodule primordia. CRISPR–Cas9-mediated knockout of the three WOX5 homologs, wox5abc mutants, reduced nodule number and attenuated nitrogenase activity, attributable to a decrease in primordium density rather than impaired rhizobial infection. Promoter dissection identified a 442 bp legume-specific promoter fragment within the WOX5a promoter that is both necessary and sufficient for primordium-specific expression. Chromatin immunoprecipitation and dual-luciferase assays revealed that this promoter fragment is directly bound by the symbiosis-responsive transcription factor NF-YAc to activate expression of WOX5a. Loss of NF-YAc phenocopied wox5abc, and NF-YAc overexpression failed to rescue nodulation in wox5abc mutants. Collectively, our findings reveal that NF-YAc-mediated activation of WOX5 initiates a de novo stem-cell niche in root cortical cells, providing the critical developmental trigger for nodule primordium initiation in soybean.

Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.

Iron is a crucial micronutrient for plants, but its availability in soil is often limited. Iron deficiency compromises plant growth, and low iron content in crops contributes substantially to the ‘hidden hunger’ that affects human health globally. The elucidation of Strategy I (reduction-based) and Strategy II (phytosiderophore-based) for iron acquisition was a milestone in plant biology and enabled the development of biofortification concepts. However, recent genetic evidence reveals that the boundary between the two strategies is blurred, with many plants possessing elements of both. Here we show that plant iron uptake mechanisms are more complex and diverse than the classical dichotomy suggests. We review evidence for this integrative view and highlight the critical role of microbial siderophores. We explain how plants access iron from microbial siderophores not only indirectly through Strategy I and II pathways but also via the direct uptake of iron–siderophore complexes, an overlooked mechanism that we introduce as Strategy III. We propose three potential routes for this direct uptake and conclude that harnessing Strategy III holds great potential for novel agricultural interventions to enhance iron biofortification and improve human health.

Ammonia (NH₃) is a critical molecule for agriculture, industry, and emerging energy applications. However, current industrial production by the Haber–Bosch process is energy-intensive and environmentally damaging, accounting for a significant portion of global CO₂ emissions. In contrast, biological nitrogen fixation (BNF) offers a sustainable alternative by converting atmospheric nitrogen (N₂) into NH₃ under ambient conditions through the action of nitrogenase enzymes. Among diazotrophic microorganisms, Azotobacter vinelandii stands out due to its ability to fix nitrogen aerobically, supported by unique physiological and genetic adaptations that protect its oxygen-sensitive nitrogenase. This review presents a comprehensive overview of NH₃ synthesis with A. vinelandii, highlighting its biochemical mechanisms, nitrogenase structure and function, and the protective strategies that enable aerobic nitrogen fixation. We further examine the diversity and advantages of Azotobacter strains, with a focus on A. vinelandii’s potential for engineered NH₃ production through synthetic biology, metabolic engineering, and emerging bio-based technologies such as photobiocatalysis and bioelectrochemistry. Recent innovations aimed at improving nitrogenase expression, cellular stability, and overall system efficiency are discussed in the context of advancing A. vinelandii as a robust chassis for industrially scalable, carbon-neutral NH₃ synthesis. The review emphasizes the importance of free-living nitrogen fixers in addressing the challenges of sustainable NH₃ production and provides insights into future directions for research and application.

Plants exist as complex holobionts, whose health and productivity depend on dynamic interactions with diverse microbial partners. This chapter explores the ecological, molecular, and biotechnological dimensions of plant-microbe relationships, tracing the continuum from natural symbioses to their translation into microbe-based agricultural applications. It examines how plants actively structure their microbial communities across distinct yet interconnected compartments, such as the rhizosphere, phyllosphere, and endosphere, and how these microorganisms in turn modulate plant physiology, nutrient acquisition, and defense. Moving beyond the study of individual strains, we discuss the growing relevance of community-level approaches and synthetic microbial consortia as frameworks to decipher and replicate the ecological principles that sustain beneficial interactions. Finally, this chapter reflects on how the integration of ecological knowledge with emerging technologies such as synthetic biology, systems modeling, and artificial intelligence offers new opportunities to design resilient, predictable, and sustainable microbe-based solutions. By learning from these naturally optimized alliances, we can guide the transition to more adaptive and sustainable agricultural systems while sustaining plant health and productivity.

Soybean plays a crucial role in meeting nitrogen demands through biological nitrogen fixation (BNF), a process highly dependent on phosphorus availability. Low-phosphorus (LP) stress significantly impairs nodule development, thereby affecting soybean growth and productivity. Genome-wide association study (GWAS) was conducted using the ratio of the nodule numbers (RNNs) under normal phosphorus condition and low-phosphorus condition in a natural population with 272 soybean accessions grown in three environments. A total of 21 novel single nucleotide polymorphisms (SNPs) related to nodule-related traits located on soybean chromosome 5 and chromosome 6 were repeatedly detected in two environments. Among them, 18 SNPs related to the ratio of the nodule number to the total plant weight under the normal phosphorus (NP) condition to that under the LP condition (RNP) formed a SNP cluster, and one SNP (AX-94275075) in this SNP cluster was detected simultaneously for multiple traits. A candidate gene, named GmbHLH135, which encodes a member of the basic helix-loop-helix (bHLH) family of transcription factors, was functionally characterized. The expression of GmbHLH135 was affected by low-phosphorus stress. Overexpressing and suppressing GmbHLH135 in soybean hairy roots resulted in a decreased and increased nodule number, respectively. GmbHLH135-overexpressing transgenic soybean lines presented decreased nodule number, brassinosteroids (BR) contents, plant biomass and yields. These findings could highlight the role of identified significant SNPs and the candidate gene GmbHLH135 in regulating nodule development under LP stress, provide valuable insights into the molecular mechanisms underlying phosphorus-mediated nodule growth and offer potential targets for soybean breeding.

Plant-microbe interactions are important for plant development, particularly mutualisms where host promiscuity allows associations with diverse microbial partners. This flexibility is crucial for adaptation, especially in invasive species. Understanding microbial community dynamics is therefore key to explain invasion success. Acacia longifolia, a member of the Fabaceae family, is an aggressive invader capable of establishing symbioses with several microorganisms including rhizobia within root nodules. These interactions promote growth and play a role in its invasive capacity. However, the nodulation process remains poorly understood, regarding microbial succession and the dynamics of these communities, following nodule development. We assessed microbial profiles in root nodules from 1-year-old saplings in two habitats using Next-Generation Sequencing, targeting 16 S and 25-28 S rRNA genes. Nodules were classified by size as a proxy for developmental stage. Our findings show that (i) different developmental stages have a characteristic microbial community; (ii) there is a shift in dominance (i.e., abundance) from early to fully developed stage, with nodules containing respectively more microbes from seed or from soil; (iii) microbial partners change in each habitat. The microbial succession indicates a shift in abundance over time, highlighting mostly the changes in recruitment: while several genera that dominate early-stage nodules are mostly found in seeds, fully developed nodules have a community mostly acquired from the surrounding soils and showed a much more specialized fungal community. Our study shows a dynamic assembly of root nodules communities within invasive range that might contribute to the plasticity and adaptative strategy of A. longifolia in these new habitats.

Accurate quantification of root nodules is essential for understanding legume–rhizobia symbiosis and improving biological nitrogen fixation. Fluorescently labeled rhizobial strains enable clear visualization of nodules; however, automated segmentation and counting remain challenging under data-limited conditions. In this study, we present a systematic benchmarking framework to evaluate three complementary approaches for fluorescent root nodule segmentation and quantification: a rule-based computer vision pipeline with optimized color-space thresholding, supervised deep learning using YOLOv12-seg transfer learning, and the training-free Segment Anything Model (SAM). Experiments were conducted on a pilot-scale dataset comprising 16 fluorescent images of Pisum sativum roots with manually annotated blue and yellow nodules. To address instability associated with small test sets, a 4-fold cross-validation strategy was employed, and performance was reported as mean ± standard deviation across folds. Model performance was evaluated using pixel-level overlap metrics (IoU, Dice), instance-level precision, recall, and F1-score, and total nodule counting error (MAE and
). The rule-based approach achieved strong segmentation and counting accuracy when fluorescence provided clear chromatic separation, demonstrating near-zero bias in total counts. Among deep learning models, YOLOv12-m provided the most balanced performance, achieving high segmentation accuracy while minimizing counting error and inter-fold variability. Larger YOLO variants did not consistently improve quantitative outcomes, suggesting overfitting under data-scarce conditions. SAM produced stable segmentation masks without training, but systematically underestimated nodule counts and lacked intrinsic class discrimination. Overall, the results highlight that segmentation fidelity alone is insufficient for reliable biological phenotyping and that accurate nodule counting must be explicitly considered. While limited in scope, this study establishes a reproducible benchmarking framework for fluorescent nodule phenotyping and provides practical guidance on method selection under constrained data and computational resources. The findings are intended as a proof-of-concept and motivate future work on larger, publicly available fluorescent datasets and hybrid segmentation strategies.

Establishing the symbiosis between legumes and nitrogen-fixing rhizobia requires the precise modulation of auxin levels. However, our understanding of auxin’s regulatory roles, particularly rhizobia-derived auxins, remains limited. Our study reveals that the auxin biosynthesis gene YUC2a is essential for the spatiotemporal control of nodule development in soybean (Glycine max). This process is orchestrated by three transcription factors: Nuclear Factor-YA9 (NF-YA9), Lateral Organ Boundaries Domain 41 (LBD41), and Nodule Inception 1a (NIN1a). In the early stages of nodulation, rhizobial auxin stimulates NF-YA9 expression, NF-YA9 then activates YUC2a expression in the cortical cell layer, establishing optimal auxin levels for nodule initiation. In the middle stages, rhizobial auxin elevates LBD41 expression, and LBD41 suppresses YUC2a to control auxin levels, ensuring proper rhizobia colonization. In the late stages, rhizobial auxin inhibits NIN1a expression, which increases YUC2a expression in nitrogen-fixing symbiosomes, fine-tuning optimal auxin levels for nodule maturation. Disruption of YUC2a and its homologs impairs cell division in nodule primordia, reducing nodule density and nitrogen fixation capacity. Conversely, cortex-specific overexpression of YUC2a promotes nodule formation but inhibits rhizobia colonization. This dynamic auxin regulation optimizes nodule development in soybean, revealing rhizobia-derived auxin's critical role in nitrogen-fixing symbiosis.