Network Walking charts transcriptional dynamics of nitrogen signaling by integrating validated and predicted genome-wide interactions

Charting a temporal path in gene networks requires linking early transcription factor (TF)-triggered events to downstream effects. We scale-up a cell-based TF-perturbation assay to identify direct regulated targets of 33 nitrogen (N)-early response TFs encompassing 88% of N-responsive Arabidopsis genes. We uncover a duality where each TF is an inducer and repressor, and in vitro cis-motifs are typically specific to regulation directionality. Validated TF-targets (71,836) are used to refine precision of a time-inferred root network, connecting 145 N-responsive TFs and 311 targets. These data are used to chart network paths from direct TF ₁ -regulated targets identified in cells to indirect targets responding only in planta via Network Walking. We uncover network paths from TGA1 and CRF4 to direct TF ₂ targets, which in turn regulate 76% and 87% of TF ₁ indirect targets in planta, respectively. These results have implications for N-use and the approach can reveal temporal networks for any biological system.