A B-ARR-mediated cytokinin transcriptional network directs hormone-cross regulation and shoot development. Arabidopsis thaliana

While the biosynthesis, degradation, and signaling pathways for the essential plant hormone cytokinin have been extensively studied, the direct transcriptional targets of B-type ARRs, the key TFs essential for cytokinin action have not been defined. We epitope tagged four B-ARRs (1, 10, 12, and 14) using recombineering techniques and profiled TF binding sites using chromatin immunoprecipitation sequencing (ChIP-seq). The resulting cytokinin transcriptional response network allowed construction of the first cytokinin TF network, identification of numerous direct downstream targets along with the B-ARR-6BA motif and elucidation of one mechanism of activation of WUSCHEL, which requires a high concentration of cytokinin in shoot apical meristem for TF binding. Overall design: we present the profiles of in vivo DNA binding sites for the genetically redundant type-B ARABIDOPSIS RESONSE REGULATORS (B-ARRs): ARR1, ARR10, and ARR12 along with ARR14 using recombineering YFP tagged lines and subsequent chromatin immunoprecipitation sequencing (ChIP-seq) analysis. The expression and genomic protein localization of B-ARRs overlap extensively, demonstrating numerous common targets among the B-ARRs. The negative regulators of cytokinin signaling, the type-A ARRs (A-ARRs), are the top ranked targets of B-ARRs. These findings confirm an immediate negative B-ARR-driven feedback loop in cytokinin signaling. Constructing a cytokinin primary response transcriptional network revealed a recurring theme involving extensive cross-regulation both within the components of the cytokinin pathway and with other plant hormone response pathways.