Comparative gene regulatory network mapping of Brassicaceae members with differential drought tolerance

Climate change is projected to intensify weather extremes like droughts that threaten global crop yields. While many crops are sensitive to water deprivation, they have drought-tolerant wild relatives; however, the molecular basis for this differential tolerance among closely related species remains unclear. We previously investigated this using transcriptomics in the Brassicaceae family, comparing the drought-sensitive reference plant Arabidopsis thaliana (Ath) with its drought-tolerant relatives Arabidopsis lyrata (Aly) and Eutrema salsugineum (Esa), uncovering key differences that may underlie their respective phenotypes. Here, to elucidate the regulatory architecture driving these differences, we mapped drought-responsive gene regulatory networks (GRNs) for all three species using the yeast one-hybrid (Y1H) approach. We cloned promoters of orthologs with divergent drought-induced expression patterns and screened them against the Ath transcription factor (TF) collection, generating a quality-controlled cross-species drought-responsive GRN map encompassing 1,137 high-confidence TF-promoter interactions. Comparative analysis revealed that the tolerant species share greater regulatory similarity and exhibit higher network connectivity than Ath. An Esa-specific expansion of bZIP TF interactions was observed, consistent with an enrichment of G-box motifs in its genome and in drought-upregulated genes. Several observed changes affect the ABA signalling pathway: Ath gained a susceptibility linked GBF3-CYP707A1 edge, whereas Esa acquired tolerance associated ABF-mTERF10 interactions. Furthermore, network-rewiring analysis uncovered a novel role for ASIL2 in stress response. Finally, our network highlights TFs (CSDP1, ERF4, HB6, and MYB73) that likely contribute to the stress-primed state of Aly and Esa. Our work provides a broadly applicable framework for comparative GRN mapping and a valuable resource for improving drought tolerance in crops.