Iron retention coupled with trade-offs in localized symbiotic effects confers tolerance to combined iron deficiency and drought in soybean

Iron (Fe) and water availability are closely interlinked, with deficiencies in both adversely affecting soybean growth. However, the strategies employed by soybean to tolerate such conditions remain poorly understood. This study elucidates the interactions of host factors, and microbial associations using multi-omics approaches in Clark (tolerant) and Arisoy (sensitive) genotypes exposed to Fe deficiency and drought. Clark exhibited resilience to stress through sustained osmotic regulation, nutrient uptake, and photosynthetic activity, in contrast to Arisoy. Particularly, Fe retention in Clark, accompanied by the upregulation of ferritin-like proteins, may mitigate oxidative stress by reducing Fenton reactions. Furthermore, higher jasmonic and salicylic acid levels in Clark may contribute to its enhanced stress adaptation compared to Arisoy. RNA-seq analysis revealed 818 and 500 upregulated, along with 931 and 361 downregulated genes, in the roots of Clark and Arisoy, respectively, under stress. We observed the upregulation of symbiotic genes, such as Chalcone-flavonone isomerase 1 and SWEET10 , accompanied by increased rhizosphere siderophore and root flavonoid in Clark. This indicates a significant role of microbes in mediating differential stress tolerance in soybean. Particularly, the combined stress led to distinct root and nodule microbiome dynamics, with Clark recruiting beneficial microbes such as Variovorax and Paecilomyces , whereas Arisoy exhibited the opposite pattern. In addition, Clark maintained nodule Bradyrhizobium and tissue nitrogen status, supported by ammonium retention and induction of Ammonium transporter 1 in the roots. Furthermore, in vitro compatibility between V. paradoxus and P. lilacinus suggests a synergistic interaction, with their localized signals benefiting Clark. Remarkably, enriched microbiomes significantly improved growth parameters, accompanied by elevated rhizosphere siderophore in sensitive genotypes under stress. This study is the first to uncover mechanisms of dual stress tolerance in soybean that may offer promising targets for breeding programs and microbiome-based biofertilizer strategies to improve combined stress tolerance in soybean and other legumes.