Defining the Role of the Cytokinin Response Factors (CRFs) in Arabidopsis thaliana

Cytokinin is a phytohormone that plays an integral role in regulating the growth, development and physiological responses of a plant. Among the processes linked to cytokinin signaling are meristem maintenance, root growth, rosette size, seed count, pathogen defense and leaf senescence. The signaling pathway consists of a hybrid two-component system consisting of the histidine kinase receptors, phosphotransfer proteins and response regulators. Numerous genes are activated or repressed downstream of the primary signaling pathway. A large number of these genes are characterized as transcription factors and therefore drive appropriate downstream gene expression in response to cytokinin. This transcriptional cascade is complex and consists of many components and feedback loops, many that have yet to be determined The Cytokinin Response Factors (CRFs) are a family of genes activated downstream of the cytokinin signaling pathway and were first identified by their induction in response to cytokinin. The CRFs are members of the AP2/ERF transcription factor family, one of the largest found in plants. Through mutant analysis, we show that the CRFs negatively regulate several cytokinin related processes, dependent on their interaction with the Arabidopsis Histidine Phosphotransfer proteins (AHPs). The CRFs positively regulate meristem size, hypocotyl elongation in the dark and the rate of leaf senescence. Additionally, through expression analyses and protein binding microarray, we were able to uncover some of the downstream processes and targets with which the CRFs are involved. We show that CRFs bind the GCC box with high affinity, as is common of other AP2/ERF members. Microarray results show the CRFs regulate genes involved in many downstream processes and also regulate some of the cytokinin signaling components. Combining both phenotypic and expression analyses, we are able to elucidate many developmental processes in which the CRFs participate, as well as to define them as negative regulators of many cytokinin regulated processes.