RESUMO
Cytokinin response factors (CRFs) are important transcription factors that form a side branch of the cytokinin signaling pathway and have been linked to cytokinin-regulated processes during development. CRF proteins are defined as belonging to a specific transcription factor family by the presence of an AP2/ERF DNA-binding domain and can be distinguished within this family by a group-specific CRF domain involved in protein-protein interactions. Here we further delimit CRFs into five distinct clades (I-V) represented across all major angiosperm lineages. Protein sequences within each clade contain a clade-specific C-terminal region distinct from other CRFs, suggesting ancient evolutionary divergence and specialization within this gene family. Conserved patterns of transcriptional regulation support these clade divisions. Despite these important differences, CRFs appear to show preferential localization or targeting to vascular tissue in quantitative real-time PCR and reporter line analyses of Arabidopsis thaliana and Solanum lycopersicum (tomato). Phloem tissue expression within the vasculature often appears the strongest in CRF reporter lines, and an analysis of CRF promoter sequences revealed conservation and significant enrichment of phloem targeting cis-elements, suggesting a potential role for CRFs in this tissue. An examination of CRF loss-of-function mutants from cytokinin-regulated clades revealed alterations in higher order vein patterning. This supports both the general link of CRFs to vascular tissue and clade-specific differences between CRFs, since alterations in vascular patterning appear to be clade specific. Together these findings indicate that CRFs are potential regulators of developmental processes associated with vascular tissues.
