Vascular Expression and C-Terminal Sequence Divergence of Cytokinin Response Factors in Flowering Plants

Abstract

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.

CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3 Regulate Lateral Root Development in Response to Cold Stress in Arabidopsis

“…The Arabidopsis CRF gene family comprises 12 members (Rashotte and Goertzen, 2010;Zwack et al, 2012). We first examined the expression profiling of CRFs using the Genevestigator Web tool, which provides a summary of gene expression responses to a variety of stimuli, in order to identify the CRF genes that are responsive to cold.…”

Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain

“…[4][5][6][7][8] While CRFs were originally identified as a handful of cytokinin regulated/associated genes in Arabidopsis and tomato, they are now known to occur in all land plants with significant diversification into distinct lineages in Angiosperms. 5,6,9,10 Two highly conserved domains are present in CRF proteins: the AP2/ERF DNA binding domain that is characteristic for all AP2/ERF proteins, and a N-terminal CRF domain. 6,11 The CRF domain is unique to CRF proteins, distinguishing CRFs from other AP2/ERFs.…”

“…12 There is also a putative serine/threonine phosphorylation site [SP(T/V)SVL] that is conserved in all CRFs. 10 The C-terminal region of the CRFs immediately following this motif is highly clade-specific in flowering plants, i.e. CRFs can be grouped into 5 distinct lineages based on sequence identity in this region.…”

“…CRFs can be grouped into 5 distinct lineages based on sequence identity in this region. 10 Although the function of these clade specific C-terminal regions is unclear it seems likely that they would be involved in lineage-specific functional roles. Experiments to test this hypothesis are ongoing and lineage specific cytokinin transcriptional regulation has been shown with Clade I, III, and IV genes being induced by cytokinin, while Clade II and V genes are not.…”

“…Experiments to test this hypothesis are ongoing and lineage specific cytokinin transcriptional regulation has been shown with Clade I, III, and IV genes being induced by cytokinin, while Clade II and V genes are not. 10 Plant transcription factors typically consist of a DNA-binding domain, an oligomerization site, a transcription activation domain (TAD) and a nuclear localization signal (NLS). 13 Activation domains function by interacting with several proteins of the basic transcription apparatus as well as a diverse group of co-activating proteins that are required for the activation of transcription but are not part of the pre-initiation complex.…”