Functional mutants of *Azospirillum brasilense* elicit beneficial physiological and metabolic responses in *Zea mays* contributing to increased host iron assimilation

The study investigates how functional mutants of the plant growth promoting bacterium Azospirillum brasilense affect iron assimilation in maize (Zea mays). Iron is abundant in soils but often poorly bioavailable, limiting plant growth and crop yield. Plants use distinct strategies to acquire iron, including proton-mediated solubilization and reduction (Strategy I) and phytosiderophore-mediated chelation and uptake (Strategy II in graminaceous plants like maize). Beneficial microbes are increasingly used to enhance plant growth and micronutrient uptake, yet their mechanisms, particularly for iron acquisition, remain insufficiently understood. The authors hypothesize that A. brasilense strains differing in biological nitrogen fixation (BNF) and auxin production capacities elicit distinct physiological and metabolic responses in maize that promote iron uptake and translocation. By leveraging functional mutants (HM053: Nif+ hyper-fixing, high auxin; FP10: Nif−, auxin-producing; ipdC: impaired in auxin biosynthesis), and employing isotopic tracers (59Fe and 11CO2), the study aims to elucidate microbial mechanisms enhancing host iron assimilation and associated host carbon allocation and metabolite changes.

Prior work has shown that plant growth promoting bacteria (PGPB) can enhance plant growth via multiple mechanisms: antagonism against phytopathogens and induction of resistance; phytostimulation through microbial production of hormones (auxin, cytokinins, gibberellins) and nitric oxide; improved nitrogen acquisition via biological nitrogen fixation; and enhanced micronutrient uptake. Azospirillum brasilense is a well-studied PGPB and prolific colonizer of grass roots, known for N2 fixation and auxin production. Field studies with the HM053 mutant have shown improved maize growth (larger stem diameter, increased leaf thickness, higher chlorophyll) and higher kernel numbers, effects previously ascribed to increased host iron uptake and improved seed iron content. Regarding iron nutrition, plants use Strategy I (proton extrusion, Fe3+ reduction to Fe2+, uptake via ZIP transporters) in non-graminaceous species and Strategy II (phytosiderophore secretion, Fe3+ chelation, uptake via YS/YSL transporters) in graminaceous plants like maize. Once absorbed, iron is transported as Fe3+-citrate complexes in xylem, and also as nicotianamine (NA), mugineic acids (MA/DMA), or as Fe2+-histidine complexes. The maize genome encodes multiple ZIP transporters, though their roles in Fe2+ uptake are not fully defined. This background sets the stage for exploring microbial influences on these iron acquisition and transport pathways.

Plant growth: Maize (Hybrid 8100) seeds were dark-germinated for 2 days at room temperature. After inoculation (as appropriate), seedlings were transplanted to growth pouches wetted with sterile Hoagland's basal salts for ~1 week, then moved to an aeroponics system with nutrients replenished every 5 days. Two-week-old plants were used for 59Fe radiotracer and ICP-MS studies. For 11C physiology/metabolism and root emission work, plants were grown in 8-inch plastic cones filled with Turface (expanded clay) with the base immersed in deionized water; Hoagland's solution was added every 3 days. Growth conditions: 12-h photoperiod, 500 µmol m−2 s−1 light, 25/20 °C day/night, 70–80% humidity for 2 weeks. Bacterial strains and inoculation: Functional mutants of A. brasilense were obtained under MTA: HM053 (natural mutant of FP2, Nif+ hyper-fixing, high auxin; selected via EDA resistance), FP10 (Nif− mutant from FP2 via N-nitrosoguanidine mutagenesis), and ipdC (site-specific disruption of indole-3-pyruvate decarboxylase gene in FP2 using SacB-cassette and DIRex recombineering, reducing auxin biosynthesis to 10% of WT). Bacteria were grown in NFbHP-lactate medium, washed, and diluted to ~10^6–10^8 CFU mL−1. Root inoculation: 1 mL inoculum added to 10 seedlings in a Petri dish, rocked 2 h, then seeds placed in germination pouches for 5 days before transplanting. 59Fe radiotracer assays: Radioactive 59Fe (t1/2 44.5 d) used as Fe2+ or Fe3+. One hour pre-administration, plants were placed in 100 mL of 5 µM Fe3+-EDTA to avoid stress; auxin co-treatment used 30 µM indole-3-acetic acid in 100 mL. 0.74 MBq 59Fe tracer added; after 3 h, roots and shoots were separated, washed (water then EDTA), blotted, and gamma-counted to calculate percent Fe assimilation and allocation. 11CO2 production and application: 11CO2 (t1/2 20.4 min) was produced on a GE PETrace cyclotron via 14N(p,α)11C from high-pressure N2 targets. Plants were pulse-labeled and incubated 3 h prior to separating load leaf, shoots, roots, and media. Roots were washed with 100 mL DI water to collect surface exudates. All components were gamma-counted with decay correction to determine total plant 11C activity, leaf export, root allocation, and root exudation fractions. Root wash aliquots were passed over AG1 X-8 anion exchange resin to separate acidic vs non-acidic exudate fractions (by counting column and breakthrough rinse). [11/12C] metabolite analyses: Separate 20-min 11CO2 exposures were followed by rapid quenching (liquid N2), tissue pulverization, and extraction in 60:40 methanol:water. Insoluble and soluble fractions were separated and counted. [14C]-Sugars were analyzed by radio-TLC on NH2-silica plates. [14C]-Amino acids were analyzed via pre-column OPA derivatization and gradient radio-HPLC. [14C]-Organic acids were isolated using QMA Sep-Pak, rinsed, and analyzed by gradient radio-HPLC with UV (210 nm) and on-line NaI detector on a Phenomenex Gemini C18 column using phosphate buffer (pH 2.5) and methanol:acetonitrile solvent system. [14C]Nicotianamine was quantified by chelation with ferrous sulfate followed by radio-HPLC (UV at 243 nm; Gemini C18; phosphate buffer pH 2.5 and methanol:acetonitrile gradient). Specific activities for [14C]histidine, [14C]citric acid, and [14C]nicotianamine were calculated as % fixed 14C activity per µmol metabolite per g fresh weight. Microbial auxin biosynthesis assay: [2-14C]indole was synthesized from [14C]HCN within 1 h at >98% radiochemical purity and SA 176 ± 24.8 GBq µmol−1. Purified tracer was incubated with washed bacterial suspensions (OD600 = 1.0 ~ 10^8 cells mL−1) up to 2 h at ambient temperature with periodic sampling. [2-14C]indole-3-acetic acid production was quantified by radio-HPLC (Gemini C18, 50:50 acetonitrile:water, 1 mL min−1, fluorescence Ex 230/Em 360 nm, NaI detector in series). Elemental analysis (ICP-MS and LA-ICP-MS): Dried roots and shoots were microwave-digested in concentrated nitric acid. Digestates were diluted and analyzed by Perkin-Elmer NexION ICP-MS in KED mode, measuring 56Fe normalized to 12C, with internal standards (Sc, In, Tl) and certified reference materials (NIST SRM 1570 and 1573). For spatial mapping, root sections (100 µm) were prepared in OCT, freeze-dried on quartz slides, and analyzed by Laser Ablation-ICP-MS. Transmission electron microscopy: Root specimens from control and inoculated plants were prepared using core facility standard protocols and imaged by TEM (details in Supplementary Methods). Root ethylene emission: Roots (from 2–3 plants per replicate) were weighed and sealed in jars for 90–130 h; 5 mL headspace samples analyzed by GC-FID (ShinCarbon ST column; 40 °C hold 2 min, ramp 10 °C min−1 to 250 °C; injector 250 °C, detector 300 °C). Emission reported as pmol ethylene gfw−1 h−1 versus treatment. Root gravitropism: Seeds grown upright in Gelrite media; upon reaching mid-depth, boxes rotated 90°. After 12 h, roots were imaged and bending angles quantified in ImageJ as an indirect indicator of auxin status, across bacterial treatments. Root indole volatile emission: Fresh roots were sealed in jars 48–90 h with airflow through Tenax GR traps (50 mg) at 50 mL min−1 for 1 h. Traps were eluted with methylene chloride and analyzed by GC-FID (RTX-WAX column; program as for ethylene). Indole retention ~15 min; emission rates reported as pmol indole gfw−1 h−1. Root DIMBOA measurements: Root tissue (700–800 mg) was extracted with water, partitioned into ethyl acetate twice, and analyzed by GC-FID (RTX-WAX; 70 °C 2 min then 10 °C min−1 to 250 °C; injector 250 °C, detector 300 °C). DIMBOA retention ~24 min; quantified against standards and reported as µmol gfw−1. In vitro chemotaxis assays for DIMBOA effects on bacterial growth were initiated using aqueous DIMBOA standards (details truncated).

- 59Fe radiotracer and ICP-MS measurements showed significant differences in maize Fe2+/Fe3+ uptake depending on the A. brasilense mutant, correlating with their biological functions (BNF capacity and auxin production). - Among the mutants, HM053 exerted the strongest effect, markedly enhancing host iron uptake. - 11CO2 tracing revealed increased allocation of newly fixed carbon to roots in HM053-treated plants, with greater transformation and exudation of 11C-labeled acidic substrates from roots that likely facilitate Fe3+ chelation in the rhizosphere. - HM053 also increased 11C partitioning into citric acid, nicotianamine, and histidine, metabolites associated with internal iron chelation and translocation (e.g., Fe3+-citrate in xylem, Fe–NA, Fe–histidine complexes). - These physiological and metabolic changes collectively support improved Fe acquisition at the root–soil interface and enhanced in planta iron transport once assimilated.

The results support the hypothesis that functional traits of A. brasilense, particularly strong BNF activity and high auxin production as in HM053, drive host physiological and metabolic shifts that enhance iron assimilation in maize. Increased allocation of new photosynthate to roots and exudation of acidic compounds would acidify and chelate Fe3+ in the rhizosphere, promoting mobilization and uptake consistent with Strategy II processes in grasses. Internally, elevated synthesis or labeling of citric acid, nicotianamine, and histidine aligns with known iron transport forms (Fe3+-citrate in xylem; Fe–NA and Fe–histidine complexes), indicating that microbial interaction not only improves acquisition but also facilitates systemic iron translocation. The differential responses among mutants link microbial auxin biosynthesis and nitrogen fixation capacities to host carbon partitioning and iron handling, providing mechanistic insight into how PGPB can enhance micronutrient nutrition and, by extension, plant growth and productivity.

This study demonstrates that specific functional mutants of Azospirillum brasilense, especially HM053, can significantly enhance iron uptake and internal translocation in maize by modulating host carbon allocation to roots, increasing exudation of acidic chelators, and augmenting metabolic pathways producing key iron-chelating compounds (citric acid, nicotianamine, histidine). The combined use of 59Fe and 11CO2 tracers with ICP-MS and metabolite profiling elucidates mechanistic links between microbial traits (BNF and auxin production) and host iron nutrition. These insights advance understanding of PGPB–plant interactions and support the development of targeted microbial inoculants to improve micronutrient acquisition and crop performance. Future work should validate these mechanisms under diverse soil conditions and field environments, dissect regulatory networks connecting microbial signals to host iron transporters and chelator biosynthesis, and explore optimization of inoculant traits for agronomic application.