Plant growth-promoting bacteria (PGPB) significantly impact plant growth and yield. Their mechanisms include pathogen antagonism, phytostimulation (hormone production), improved nitrogen uptake (BNF), and enhanced micronutrient uptake. *Azospirillum brasilense*, a prominent grass root colonizer, is a well-studied PGPB known for nitrogen fixation and auxin production. Previous work demonstrated that maize inoculated with the *A. brasilense* mutant HM053 showed increased stem diameter, leaf thickness, chlorophyll content, and crop yields, attributed to enhanced iron uptake. Iron is essential for plant growth, yet its bioavailability is limited in aerobic, neutral pH soils due to its insolubility as Fe³⁺ oxyhydroxides. Iron deficiency reduces plant growth and yield, while excess iron causes cellular damage. Plants employ two distinct strategies for iron uptake: Strategy I (reduction-based) in non-graminaceous plants and Strategy II (chelation-based) in graminaceous plants like maize. This study aims to elucidate the mechanisms by which *A. brasilense* enhances iron uptake in maize using functional mutants with varying capacities for nitrogen fixation and auxin production. This understanding is crucial for developing sustainable agricultural practices.

Extensive research demonstrates the significant influence of PGPB on plant growth and yield. Mechanisms include antagonism against plant pathogens [1], phytostimulation through hormone production [2, 3], improved nitrogen uptake via biological nitrogen fixation (BNF) [4], and enhanced micronutrient uptake [5, 6]. *Azospirillum brasilense*, a diazotrophic bacterium, is a well-known PGPB that colonizes grass roots, fixing nitrogen and producing auxin [3, 4]. Commercial inoculants of *A. brasilense* have shown promising results in field trials with various grain crops [7-9]. The importance of iron in plant growth is well-established; however, its bioavailability is often limited in many soils. Plants have evolved mechanisms to acquire iron, differing between graminaceous and non-graminaceous species [11-35]. While the potential of beneficial microbes to improve plant micronutrient uptake is compelling, the underlying mechanisms remain largely unclear.

This study utilized functional mutants of *A. brasilense* (HM053, FP10, *ipdC*) with varying capacities for nitrogen fixation and auxin production. Maize seeds were inoculated with these mutants and grown in aeroponic and Turface™ systems. Radioactive ⁵⁹Fe (Fe²⁺ and Fe³⁺) was used to trace iron uptake, and ICP-MS measured total iron content. Radioactive ¹¹CO₂ was used to study carbon allocation and metabolism. Root exudates were analyzed to determine acidic and non-acidic fractions. ¹¹C-metabolite analysis included sugars, amino acids, organic acids, nicotianamine, and histidine. Auxin biosynthesis was assessed using [2-¹⁴C]indole. Additional analyses included laser ablation-ICP-MS, transmission electron microscopy, root ethylene emission, root gravitropism, root indole measurements, and root DIMBOA measurements. *In vitro* chemotaxis assays examined the effects of DIMBOA on bacterial growth.

Significant differences in host iron uptake were observed among the *A. brasilense* mutants, correlating with their biological functions. HM053, a hyper-nitrogen-fixing and high auxin-producing mutant, exhibited the greatest influence on host Fe uptake. ¹¹C-tracing revealed that HM053 significantly increased plant allocation of carbon resources to roots, leading to enhanced exudation of ¹¹C-acidic substrates that aid in iron chelation. Furthermore, HM053 increased ¹¹C partitioning into citric acid, nicotianamine, and histidine, which are crucial for iron translocation within the plant. These findings suggest that HM053 promotes iron uptake in maize through a multifaceted mechanism involving increased carbon allocation to the roots, enhanced chelation, and efficient translocation of iron within the plant.

The results demonstrate a strong correlation between *A. brasilense* mutant function and its impact on host iron uptake in maize. HM053's superior effect on iron uptake is likely due to its combined ability to enhance both nitrogen fixation and auxin production. The increased carbon allocation to the roots observed with HM053 indicates a metabolic shift towards iron acquisition. The enhanced production of ¹¹C-labeled citric acid, nicotianamine, and histidine suggests that HM053 promotes the efficient translocation of assimilated iron from the roots to the shoots. These findings support the hypothesis that beneficial microbes can enhance plant nutrient uptake through complex physiological and metabolic mechanisms. The study highlights the potential of using *A. brasilense* mutants as a tool to improve plant nutrition and crop yields.

This study demonstrates that functional mutants of *A. brasilense* can significantly enhance iron uptake in maize through complex metabolic and physiological mechanisms. HM053 stands out as a particularly effective mutant, boosting iron uptake by increasing carbon allocation to roots, enhancing chelation, and improving translocation. This research provides valuable insights into plant-microbe interactions and their potential for sustainable agriculture. Future research could focus on identifying the specific genes responsible for the observed effects and exploring the potential of these beneficial microbes in various agricultural settings.

The study was conducted under controlled environmental conditions. The results may not fully reflect the complexity of field conditions, where various environmental factors can influence plant-microbe interactions. Further studies are needed to validate these findings under diverse field conditions. The study focused primarily on iron uptake; further investigation is required to understand the effects of these mutants on other essential nutrients.