Transcriptional Dynamics of Nitrogen Fixation and Senescence in Soybean Nodules: A Dual Perspective on Host and <i>Bradyrhizobium</i> Regulation

The soybean-<i>Bradyrhizobium</i> symbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N fertilizers. However, nitrogen fixation is transient, peaking several weeks after <i>Bradyrhizobium</i> 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 <i>Bradyrhizobium diazoefficiens</i> USDA110. Weekly nodule samples (2 to 10 weeks postinoculation, wpi) were analyzed using RNA and small RNA sequencing, and acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2 to 3 wpi), nitrogen fixation (3 to 8 wpi), and senescence (8 to 10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereas <i>Bradyrhizobium</i> 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 <i>lipoxygenases</i> (<i>Lox</i>), suggesting roles for oxylipin metabolism. Soy <i>hemoglobin-2</i> (<i>Hb2</i>), previously classified as nonsymbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of the <i>Bradyrhizobium paa</i> operon and <i>rpoH</i> during senescence suggesting metabolic adaptation for survival beyond symbiosis. Additionally, <i>Bradyrhizobium nif</i> gene expression showed stage-specific regulation, with <i>nifK</i> peaking at 2 wpi, <i>nifD</i> and <i>nifA</i> at 2 and 10 wpi, and <i>nifH</i>, <i>nifW</i>, and <i>nifS</i> 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. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.