TEM analysis of iron nanoparticle-loaded endophytic bacteria mitigating salinity-induced root stress effects in maize

Salinity stress is a major abiotic constraint limiting crop productivity through osmotic imbalance, ionic toxicity, oxidative damage, and structural disruption. Here, we report a novel nano-enabled microbial cell factory system based on iron nanoparticle (FeNP)-loaded plant growth-promoting endophytic bacteria (PGPEB) isolated from Zea mays roots and identified as Bacillus sp. (16S rRNA accession MZ700077.1; 99.41% similarity). This living nano-bio hybrid was designed to integrate microbial plant growth-promoting functions with nanoparticle-mediated micronutrient delivery for enhanced salinity stress tolerance. Transmission electron microscopy confirmed successful formation of the microbial cell factory, revealing rod-shaped bacterial cells with intact flagella and uniform spherical FeNPs (~ 29.9 nm) strongly associated with the bacterial surface, indicating stable nano-bio interfacing without compromising cellular integrity. The engineered FeNP-PGPEB system significantly improved maize seed germination, root and shoot growth, and overall seedling vigor under moderate to severe salinity (200 mM NaCl), outperforming either PGPEB or FeNPs alone and demonstrating clear synergistic effects. Physiological and biochemical analyses revealed that the nano-bio formulation enhanced antioxidant defense systems (SOD, CAT, POD), increased proline accumulation, and optimized phytohormonal balance by elevating IAA and moderating ABA levels. In addition, lipid peroxidation (MDA) was reduced, while relative water content was maintained, indicating improved redox homeostasis and osmotic regulation. Ultrastructural and anatomical observations further confirmed reduced plasmolysis, improved membrane integrity, and enhanced vascular and root tissue organization under salt stress. Multivariate analyses (PCA and clustering) clearly distinguished treatments, with FeNP-PGPEB strongly associated with stress resilience traits and improved physiological stability. Overall, this study establishes a novel microbial cell factory platform, where endophytic Bacillus acts as a living nanocarrier for iron delivery, integrating microbial biotechnology and nanotechnology into a unified strategy for enhancing crop tolerance to salinity stress and advancing sustainable agricultural systems.