Transcriptional Dynamics of Nitrogen Fixation and Senescence in Soybean Nodules: A Dual Perspective on Host and

The Soybean-Bradyrhizobium symbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N-fertilizers. However, nitrogen fixation is transient, peaking several weeks after Bradyrhizobium colonization and declining as nodules senesce in coordination with host development. To investigate the regulatory mechanisms governing SNF and senescence, we conducted a temporal transcriptomic analysis of soybean nodules colonized with Bradyrhizobium diazoefficiens USDA110. Weekly nodule samples (2-10 weeks post-inoculation, wpi) were analyzed using RNA and small RNA sequencing, while acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2-3 wpi), nitrogen fixation (3-8 wpi), and senescence (8-10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereas Bradyrhizobium underwent major transcriptional shifts early in development before stabilizing during nitrogen fixation. We identified seven soybean genes and several microRNAs as candidate biomarkers of nitrogen fixation, including lipoxygenases (Lox), suggesting roles for oxylipin metabolism. Soy hemoglobin-2 (Hb2), previously classified as non-symbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of the Bradyrhizobium paa operon and rpoH during senescence suggested metabolic adaptation for survival beyond symbiosis. Additionally, Bradyrhizobium NIF gene expression showed stage-specific regulation, with nifK peaking at 2 wpi, nifD and nifA at 2 and 10 wpi, and nifH, nifW, and nifS at 10 wpi. These findings provide insights into SNF regulation and nodule aging, revealing temporal gene expression patterns that could inform breeding or genetic engineering strategies to enhance nitrogen fixation in soybeans and other legume crops.