{"paper_id":"e6af3ffb-072a-4bf6-be31-c9770e5ad7bc","body_text":"Abstract\nClimate 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.\nCompeting Interest Statement\nThe authors have declared no competing interest.","source_license":"CC-BY-4.0","license_restricted":false}