Cis-Regulatory Editing of Peptide Signaling Rewires Plant Root Architecture

Precise cis-regulatory control of gene expression is essential for plant growth. In Arabidopsis thaliana, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth-stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)-promoter interactions for nine PSY and three PSYR genes by combining high-throughput enhanced yeast one-hybrid screening with DNA Affinity Purification sequencing (DAP-seq) data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence shoot and root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We identified a CRF10-binding motif in the PSYR3 promoter using DAP-seq data and validated it using eY1H. Guided by these insights, we used CRISPR/Cas9-mediated promoter editing to delete a small region encompassing or flanking a functional TF-binding site (TFBS). Removal of this motif, or of its surrounding region, reduced PSYR3 expression and enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant. Together, our results define the global TF-promoter interaction network of the PSYR/PSY pathway and identify CRF10 as a key transcriptional regulator of PSYR3-mediated signaling. More broadly, our work demonstrates that targeting cis-regulatory regions can modulate gene expression and associated growth traits, suggesting a TFBS-focused strategy to influence plant growth patterns.