Cytokinin response factor 1 acts as a negative regulator of cytokinin-mediated developmental pathways in Arabidopsis

CRF1 functions as a negative regulator of cytokinin signaling in Arabidopsis at least in part by antagonizing ARR12-dependent SHY2 transactivation, thereby influencing primary root growth, rosette development, and leaf senescence. Cytokinin Response Factors (CRFs) are members of the AP2/ERF family of transcription factors that act downstream of the two-component cytokinin signaling pathway to regulate diverse developmental processes. In this study, we identify CRF1 as a negative regulator of cytokinin signaling in Arabidopsis thaliana. Loss-of-function crf1 mutants exhibit pronounced hypersensitivity to cytokinin, characterized by stronger inhibition of primary root growth, larger rosette size, and delay in dark-induced leaf senescence, compared to wild type. Conversely, when CRF1 was overexpressed (CRF1-OX), it impaired cytokinin-induced responses in both primary root and shoot. These phenotypic alterations in response to cytokinin correlated with transcriptional changes. At the transcript level, primary cytokinin-inducible genes, like type-A and type-B ARRs, and secondary responsive genes like SHY2, were significantly upregulated in crf1 mutants in response to cytokinin, compared to wild type. SHY2 is a known Aux/IAA protein that works to repress auxin signaling, especially in primary roots. Interestingly, while the type-B ARR, ARR12, is known to transactivate SHY2 promoter, our results show that its activity is impaired in presence of CRF1. Transactivation assays clearly indicated that ARR12 could not activate SHY2 promoter when present along with CRF1, implying that CRF1 intercepts ARR12-mediated transcriptional regulation. CRF1 thus acts as a negative regulator of cytokinin signaling, acting at least in part by antagonizing ARR12-mediated gene expression. In summary, CRF1 modulates key developmental processes in Arabidopsis by regulating cytokinin responses in both primary root and shoot, and may serve as a potential target for fine-tuning plant growth and development.