Protocols for Chromatin Immunoprecipitation Sequencing and qPCR to Investigate Pytohormone-Responsive Transcriptional Regulation in Plants.

Plant hormones regulate a wide array of developmental and stress-response pathways by modulating gene expression through specific transcription factors (TFs). Understanding how these TFs interact with their genomic targets is essential for dissecting hormone signaling networks. Chromatin immunoprecipitation (ChIP), coupled with quantitative PCR (ChIP-qPCR) or next-generation sequencing (ChIP-seq), offers a powerful approach to study in vivo protein-DNA interactions. However, ChIP in plants poses unique technical challenges due to the presence of rigid cell walls, abundant secondary metabolites, and chromatin accessibility constraints. Here, we present a detailed and optimized protocol for performing ChIP-qPCR and ChIP-seq in Arabidopsis thaliana to investigate hormone-responsive gene regulation. The protocol includes steps for hormone treatment, tissue fixation, chromatin extraction, immunoprecipitation using specific antibodies, reverse crosslinking, DNA purification, and downstream quantification. This method enables high-resolution mapping of transcription factor occupancy at target loci and can be adapted for different hormones and plant species. Compared to indirect transcriptomic approaches, ChIP-based assays provide direct evidence of TF-DNA binding and allow identification of primary targets in signaling cascades. The protocol supports mechanistic studies of hormone action in plants and facilitates the construction of gene regulatory networks.