The evolution of cytokinin signaling and its role in development before Angiosperms.

Cytokinin signaling by way of a multistep two-component signaling pathway has been highly examined over the last 20 years with a lot of attention to how its signaling components regulate essential growth and developmental processes from seedlings to senescence. Most studies have focused on Angiosperm plants, particularly Arabidopsis, with very little attention across the rest of the plant kingdom, making it difficult to know if cytokinin-regulated developmental processes are have remained evolutionarily conserved or distinct among different plant groups. Here the prevalence of cytokinin signaling components throughout plants, algae, and other organisms are examined. Particular examination is paid to how minimal sets of these base signaling components are related to development in Bryophytes, a primary group of study outside of Angiosperms. General comparisons of the role of signaling components between these distant groups suggest some evolutionary conservation of function has been maintained across plants as a whole. This review also highlights the need to investigate how cytokinin signaling components, recently identified in every plant group, contribute to development; this will give higher resolution to our understanding of cytokinin roles in development across plant taxa. Also revealed is a strong need to further pursue how cytokinin signaling components recently identified in every plant group contribute to development in order to further dissect these connections.

CYTOKININ RESPONSE FACTOR 2 is involved in modulating the salt stress response.

Cytokinin has strong connections to development and a growing role in the abiotic stress response. Here we show that CYTOKININ RESPONSE FACTOR 2 (CRF2) is additionally involved in the salt (NaCl) stress response. CRF2 promoter-GUS expression indicates CRF2 involvement in the response to salt stress as well as the previously known cytokinin response. Interestingly, CRF2 mutant seedlings are quite similar to the wild type (WT) under non-stressed conditions yet have many distinct changes in response to salt stress. Cytokinin levels measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) that increased in the WT after salt stress are decreased in crf2, potentially from CRF2 regulation of cytokinin biosynthesis genes. Ion content measured by inductively coupled plasma optical emission spectrometry (ICP-OES) was increased in the WT for Na, K, Mn, Ca and Mg after salt stress, whereas the corresponding Ca and Mg increases are lacking in crf2. Many genes examined by RNA-seq analysis were altered transcriptionally by salt stress in both the WT and crf2, yet interestingly approximately one-third of salt-modified crf2 transcripts (2655) showed unique regulation. Different transcript profiles for salt stress in crf2 compared with the WT background was further supported through an examination of co-expressed genes by weighted gene correlation network analysis (WGCMA) and principal component analysis (PCA). Additionally, Gene Ontology (GO) enrichment terms found from salt-treated transcripts revealed most photosynthesis-related terms as only being affected in crf2, leading to an examination of chlorophyll levels and the efficiency of photosystem II (via the ratio of variable fluorescence to maximum fluorescence, Fv /Fm ) as well as physiology after salt treatment. Salt stress-treated crf2 plants had both reduced chlorophyll levels and lower Fv /Fm values compared with the WT, suggesting that CRF2 plays a role in the modulation of salt stress responses linked to photosynthesis.

Cytokinin Response Factor 9 Represses Cytokinin Responses in Flower Development.

A multi-step phosphorelay system is the main conduit of cytokinin signal transduction. However, several groups of additional factors that also play a role in this signaling pathway have been found-among them the Cytokinin Response Factors (CRFs). In a genetic screen, CRF9 was identified as a regulator of the transcriptional cytokinin response. It is mainly expressed in flowers. Mutational analysis indicates that CRF9 plays a role in the transition from vegetative to reproductive growth and silique development. The CRF9 protein is localized in the nucleus and functions as a transcriptional repressor of Arabidopsis Response Regulator 6 (ARR6)-a primary response gene for cytokinin signaling. The experimental data suggest that CRF9 functions as a repressor of cytokinin during reproductive development.

Clade III cytokinin response factors share common roles in response to oxidative stress responses linked to cytokinin synthesis.

Cytokinin response factors (CRFs) are transcription factors that are involved in cytokinin (CK) response, as well as being linked to abiotic stress tolerance. In particular, oxidative stress responses are activated by Clade III CRF members, such as AtCRF6. Here we explored the relationships between Clade III CRFs and oxidative stress. Transcriptomic responses to oxidative stress were determined in two Clade III transcription factors, Arabidopsis AtCRF5 and tomato SlCRF5. AtCRF5 was required for regulated expression of >240 genes that are involved in oxidative stress response. Similarly, SlCRF5 was involved in the regulated expression of nearly 420 oxidative stress response genes. Similarities in gene regulation by these Clade III members in response to oxidative stress were observed between Arabidopsis and tomato, as indicated by Gene Ontology term enrichment. CK levels were also changed in response to oxidative stress in both species. These changes were regulated by Clade III CRFs. Taken together, these findings suggest that Clade III CRFs play a role in oxidative stress response as well as having roles in CK signaling.

Eggplant Germination is Promoted by Hydrogen Peroxide and Temperature in an Independent but Overlapping Manner.

Hydrogen peroxide promotes seed germination, but the molecular mechanisms underlying this process are unclear. This study presents the results of eggplant (Solanum melongena) germination analyses conducted at two different temperatures and follows the effect of hydrogen peroxide treatment on seed germination and the seed proteome. Hydrogen peroxide was found to promote eggplant germination in a way not dissimilar to that of increased temperature stimuli. LC-MS profiling detected 729 protein families, 77 of which responded to a temperature increase or hydrogen peroxide treatment. These differentially abundant proteins were found to be involved in a number of processes, including protein and amino acid metabolism, carbohydrate metabolism, and the glyoxylate cycle. There was a very low overlap between hydrogen peroxide and temperature-responsive proteins, highlighting the differences behind the seemingly similar outcomes. Furthermore, the observed changes from the seed proteome indicate that hydrogen peroxide treatment diminished the seed endogenous hydrogen peroxide pool and that a part of manifested positive hydrogen peroxide effect might be related to altered sensitivity to abscisic acid.

Cytokinin Response Factor 6 Represses Cytokinin-Associated Genes during Oxidative Stress.

Cytokinin is a phytohormone that is well known for its roles in numerous plant growth and developmental processes, yet it has also been linked to abiotic stress response in a less defined manner. Arabidopsis (Arabidopsis thaliana) Cytokinin Response Factor 6 (CRF6) is a cytokinin-responsive AP2/ERF-family transcription factor that, through the cytokinin signaling pathway, plays a key role in the inhibition of dark-induced senescence. CRF6 expression is also induced by oxidative stress, and here we show a novel function for CRF6 in relation to oxidative stress and identify downstream transcriptional targets of CRF6 that are repressed in response to oxidative stress. Analysis of transcriptomic changes in wild-type and crf6 mutant plants treated with H2O2 identified CRF6-dependent differentially expressed transcripts, many of which were repressed rather than induced. Moreover, many repressed genes also show decreased expression in 35S:CRF6 overexpressing plants. Together, these findings suggest that CRF6 functions largely as a transcriptional repressor. Interestingly, among the H2O2 repressed CRF6-dependent transcripts was a set of five genes associated with cytokinin processes: (signaling) ARR6, ARR9, ARR11, (biosynthesis) LOG7, and (transport) ABCG14. We have examined mutants of these cytokinin-associated target genes to reveal novel connections to oxidative stress. Further examination of CRF6-DNA interactions indicated that CRF6 may regulate its targets both directly and indirectly. Together, this shows that CRF6 functions during oxidative stress as a negative regulator to control this cytokinin-associated module of CRF6-dependent genes and establishes a novel connection between cytokinin and oxidative stress response.

Cytokinin response factor 4 (CRF4) is induced by cold and involved in freezing tolerance.

KEY MESSAGE: Cytokinin response factor 4 (CRF4) shows a short-term induction by cold (4 °C) that appears to play a role in non-acclimated freezing tolerance as seen in mutant and overexpression lines. Responses to abiotic stresses, such as cold stress, are critical to plant growth and optimal production. Examination of Arabidopsis cytokinin response factors (CRFs) showed transcriptional induction after exposure to cold (4 °C). In particular, CRF4 was strongly induced in both root and shoot tissues. As CRF4 is one of several CRFs not transcriptionally regulated by cytokinin, we further investigated its response to cold. Peak CRF4 induction occurred 6 h post cold exposure, after which expression was maintained at moderately elevated levels during extended cold and subsequent treatment recovery. Examination of CRF4 mutant and overexpression lines under standard (non-cold) conditions revealed little difference from WT. One exception was a small, but significant increase in primary root growth of overexpression plants (CRF4OX). Under cold conditions, the only phenotype observed was a reduction in the rate of germination of CRF4OX seeds. The pattern of CRF4 expression along with the lack of strong phenotype at 4 °C led us to hypothesize that cold induction of CRF4 could play a role in short-term cold acclimation leading to increased freeze tolerance. Examination of CRF4OX and crf4 plants exposed to freezing temperatures revealed mutants lacking expression of CRF4 were more sensitive to freezing, while CRF4OXs with increased levels CRF4 levels were more tolerant. Altered transcript expression of CBF and COR15a cold signaling pathway genes in crf4 mutant and overexpression lines suggest that CRF4 may be potentially connected to this pathway. Overall this indicates that CRF4 plays an important role in both cold response and freezing stress.

Interactions between cytokinin signalling and abiotic stress responses.

Plants have evolved elaborate mechanisms for sensing and responding to sub-optimal environmental conditions. Abiotic stresses caused by these conditions trigger a wide range of local and long-distance signals which must be co-ordinated and integrated into whole-plant processes, such as development, in order for the plant to respond properly and survive. Several hormones function as key regulators of stress tolerance, connecting local stimuli to systemic responses. Cytokinin is a hormone well known for its role in numerous aspects of growth and development, although abundant evidence also indicates that cytokinin functions in stress responses as well. At present, a full understanding of the effects of cytokinin on plant resistance to stress is lacking, possibly as a result of the complex interactions between cytokinin and stress signalling. Current knowledge of the physiological relationship between cytokinin and abiotic stress, based on measurements of cytokinin levels under stress conditions and the effects of cytokinin treatment on stress tolerance, has been examined here. A pattern of transcriptional regulation of stress-related genes by cytokinin in different plant species has also been identified. In addition, research regarding the role of specific cytokinin signalling components in a variety of stress responses is presented. We discuss what this body of research collectively implies with regard to cross-talk between cytokinin and abiotic stress tolerance.

Integrated signaling networks in plant responses to sedentary endoparasitic nematodes: a perspective.

Sedentary plant endoparasitic nematodes can cause detrimental yield losses in crop plants making the study of detailed cellular, molecular, and whole plant responses to them a subject of importance. In response to invading nematodes and nematode-secreted effectors, plant susceptibility/resistance is mainly determined by the coordination of different signaling pathways including specific plant resistance genes or proteins, plant hormone synthesis and signaling pathways, as well as reactive oxygen signals that are generated in response to nematode attack. Crosstalk between various nematode resistance-related elements can be seen as an integrated signaling network regulated by transcription factors and small RNAs at the transcriptional, posttranscriptional, and/or translational levels. Ultimately, the outcome of this highly controlled signaling network determines the host plant susceptibility/resistance to nematodes.

Characterization of two tomato AP2/ERF genes, SlCRF1 and SlCRF2 in hormone and stress responses.

KEY MESSAGE: SlCRF1 and SlCRF2 are expressed throughout the plant, prominently in vascular tissue. Each SlCRF has a distinct pattern of cytokinin induction and regulation by abiotic stresses in different organs. Cytokinin is an essential plant hormone involved in the regulation of many growth and developmental processes. While many cytokinin signaling pathway components have been well characterized, the cytokinin response factors (CRFs) that form a branch of this pathway are less well understood. This study examines the tomato (Solanum lycopersicum (L.)) CRF genes, SlCRF1 and SlCRF2 presenting a detailed and novel characterization of their developmental expression patterns, transcriptional regulation by hormones particularly cytokinin, and response to abiotic stresses. Both SlCRF1 and SlCRF2 were predominantly expressed in vasculature in tissues throughout the plant, with an overall trend for greater SlCRF2 expression in younger organs. Hormone regulation of SlCRF1 and SlCRF2 transcripts is primarily by cytokinin, which induced both SlCRFs in different organs over a range of developmental stages. The strongest cytokinin induction was found in leaves, with SlCRF2 induced to a higher level than SlCRF1. Examination of SlCRF transcripts during abiotic stress responses revealed that SlCRF1 and SlCRF2 have distinct patterns of regulation from each other and between leaves and roots. Novel connections between SlCRFs and stresses were found in particular including a strong induction of SlCRF1 by cold stress and a strong induction of SlCRF2 by oxidative stress in roots and unique patterns of induction/repression linking both SlCRFs to drought stress and response during recovery. Overall, this study provides a clear picture of SlCRF1 and SlCRF2 expression patterns across tissues during development and in response to cytokinin and specific stresses, indicating their importance in plant growth and environmental responses.

Citrullus colocynthis NAC transcription factors CcNAC1 and CcNAC2 are involved in light and auxin signaling.

KEY MESSAGE: Two novel NAC transcription factors from C itrullus colocynthis implicated in light and auxin signaling pathway. NAC transcription factors (NAM, ATAF1, 2, CUC2) have multiple functions in plant growth and development. Two NACs, CcNAC1 and CcNAC2, were recently identified in the highly drought-tolerant cucurbit species, Citrullus colocynthis. This study examines the functional role of these genes under different qualities of light based on the in silico analysis of the CcNAC1 and CcNAC2 promoters that revealed the presence of several light-associated motifs. The impact of both light and auxin on CcNAC1 and CcNAC2 expression was examined in C. colocynthis leaves, and using reporter (pCcNAC1, 2::GUS) lines in Arabidopsis. Furthermore, the effects of constitutive overexpression (OE-CcNAC1, 2) in Arabidopsis were also examined under a range of conditions to confirm reporter line linkages. White, blue, red, and far-red light treatments resulted in similar patterns of quantitative changes in CcNAC1and CcNAC2 expression in both species, with the highest transcript increases following red light. Photomorphogenic changes in Arabidopsis hypocotyls were correlated with gene transcript levels. In the absence of light, hypocotyls of OE-CcNAC1/CcNAC2 lines were significantly longer as compared to WT. The addition of exogenous auxin (+IAA) to growth medium also resulted in changes to the hypocotyl lengths of overexpression lines and spatiotemporal reporter line changes in seedlings. Our data suggest that CcNAC1, 2 might be functionally important in the light signaling pathway, and appear connected to the hormone auxin. This is the first study to indicate that NAC genes might play a role in both light and auxin signaling pathways.

Cytokinin response factor 6 negatively regulates leaf senescence and is induced in response to cytokinin and numerous abiotic stresses.

Cytokinin response factor 6 (CRF6) is an Arabidopsis AP2/ERF transcription factor which is transcriptionally induced by cytokinin. Cytokinin is known to delay leaf senescence in wild-type (WT) plants, for example in dark-incubated detached leaves. This response is mediated by the cytokinin receptor Arabidopsis histidine kinase receptor 3 (AHK3). Similar to ahk3 mutants, crf6 leaves show decreased sensitivity to this cytokinin effect. Leaves overexpressing CRF6 retain more Chl than those of the WT under these conditions without exogenous cytokinin. It therefore appears that an increase in expression of CRF6 downstream of the perception of cytokinin by AHK3 is involved in the delay of leaf senescence. Intact crf6 plants also begin to undergo monocarpic senescence sooner than WT plants. Interestingly, plants overexpressing CRF6 display a more extreme acceleration of development than crf6 mutants, suggesting that a specific expression level or localization of CRF6 is necessary to prevent premature senescence. Expression analyses indicate that CRF6 is highly expressed in the veins of mature leaves and that this expression decreases with age. CRF6 expression is shown to be induced by abiotic stress, in addition to increased cytokinin. Together, these findings suggest that CRF6 functions to regulate developmental senescence negatively and may have a similar role in response to stress. CRF6 may therefore be involved in fine-tuning the timing of developmental and stress-induced senescence. CRF6 functioning in negative regulation of senescence is significant in that it is the first process known to be regulated by cytokinin, in which a CRF can be placed specifically downstream of the cytokinin signaling pathway.

Transcriptome profiling of cytokinin and auxin regulation in tomato root.

Tomato is a model and economically important crop plant with little information available about gene expression in roots. Currently, there have only been a few studies that examine hormonal responses in tomato roots and none at a genome-wide level. This study examined the transcriptome atlas of tomato root regions (root tip, lateral roots, and whole roots) and the transcriptional regulation of each root region in response to the plant hormones cytokinin and auxin using Illumina RNA sequencing. More than 165 million 1×54 base pair reads were mapped onto the Solanum lycopersicum reference genome and differential expression patterns in each root region in response to each hormone were assessed. Many novel cytokinin- and auxin-induced and -repressed genes were identified as significantly differentially expressed and the expression levels of several were confirmed by qPCR. A number of these regulated genes represent tomato orthologues of cytokinin- or auxin-regulated genes identified in other species, including CKXs, type-A RRs, Aux/IAAs, and ARFs. Additionally, the data confirm some of the hormone regulation studies for recently examined genes in tomato such as SlIAAs and SlGH3s. Moreover, genes expressed abundantly in each root region were identified which provide a spatial distribution of many classes of genes, including plant defence, secondary metabolite production, and general metabolism across the root. Overall this study presents the first global expression patterns of hormone-regulated transcripts in tomato roots, which will be functionally relevant for future studies directed towards tomato root growth and development.

Vascular expression and C-terminal sequence divergence of cytokinin response factors in flowering plants.

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.

Solanum lycopersicum cytokinin response factor (SlCRF) genes: characterization of CRF domain-containing ERF genes in tomato.

Cytokinin is an influential hormone in growth and developmental processes across many plant species. While several cytokinin-regulated genes have been well characterized in Arabidopsis, few have been identified in tomato, Solanum lycopersicum. Here a tomato family of 11 highly related cytokinin response factor genes designated as SlCRF1-SlCRF11 (Solanum lycopersicum cytokinin response factor) are identified and characterized. SlCRFs are AP2/ERF transcription factors and generally orthologous to Arabidopsis CRF clade members (AtCRFs). Some SlCRF genes lack a direct Arabidopsis orthologue and one SlCRF has a unique protein domain arrangement not seen in any other CRF protein. Expression analysis of SlCRF1-SlCRF11 revealed differential patterns and levels across plant tissues examined (leaf, stem, root and flower). Several SlCRFs show induction by cytokinin to various degrees, similar to AtCRFs. Additionally it is shown that some SlCRFs can be regulated by other factors, including NaCl, ethylene, methyl jasmonate, and salicylic acid. Examination of SlCRF proteins in transient Agrobacterium infiltration experiments indicates they can be nuclear localized in planta. Using a bimolecular fluorescence complementation (split-yellow fluorescent protein) system, it is also shown that SlCRF proteins can interact to form homo- and heterodimers. Overall this work indicates that some SlCRFs resemble previously identified CRFs in terms of structure, expression, and cytokinin regulation. However, SlCRFs have novel CRF protein forms and responses to abiotic factors, suggesting they may have a diverse set of roles in stress and hormone regulation in tomato.

Advances in upstream players of cytokinin phosphorelay: receptors and histidine phosphotransfer proteins.

Cytokinins are a class of plant hormones that have been linked to numerous growth and developmental aspects in plants. The cytokinin signal is perceived by sensor histidine kinase receptors and transmitted via histidine phosphotransfer proteins (HPts) to downstream response regulators. Since their discovery, cytokinin receptors have been a focus of interest for many researchers. Ongoing research on these transmembrane receptors has greatly broadened our knowledge in terms of cytokinin-receptor interaction, receptor specificity, receptor cellular localization, and receptor functions in cytokinin related growth and developmental processes. This review focuses on the recent advances on the cytokinin receptors and HPt proteins in Arabidopsis.

Down-stream components of cytokinin signaling and the role of cytokinin throughout the plant.

Cytokinins constitute a class of plant hormones influencing numerous aspects of growth and development. These processes occur through the downstream components of the cytokinin signaling pathway after its perception and signal transduction. The importance of these downstream signaling components has been revealed through the use of both traditional genetic and advanced molecular approaches studying mutants and transgenic lines involving cytokinin and diverse plant growth and developmental processes. Interestingly, these effects are not always directly via cytokinin, but by interactions with other plants hormones or transcription factor cascades, which can involve regulatory loops that affect transcription as well as hormone concentrations. This review covers recent advancements in understanding the role of cytokinin via its signaling components, specifically the downstream responses regulators in controlling vital plant growth and developmental processes.

CRFs form protein-protein interactions with each other and with members of the cytokinin signalling pathway in Arabidopsis via the CRF domain.

Cytokinin is a plant hormone essential for growth and development. The elucidation of its signalling pathway as a variant of the bacterial two-component signalling system (TCS) has led to a better understanding of how this hormone is involved in general plant processes. A set of cytokinin-regulated transcription factors known as cytokinin response factors (CRFs) have been described as a potential branch emanating from the TCS, yet little is known about how CRFs actually interact with each other and with members of the TCS pathway. Here the interactions of CRF proteins (CRF1-CRF8) using the yeast two-hybrid system and bimolecular fluorescence complementation in planta assays are described. It was found that CRFs are readily able to form both homo- and heterodimers with each other. The first analysis of CRF versus TCS pathway protein interactions is also provided, which indicates that CRFs (CRF1-CRF8) are able specifically to interact directly with most of the Arabidopsis histidine-phosphotransfer proteins (AHP1-AHP5) further solidifying their link to the cytokinin signalling pathway. In addition, the region of CRF proteins involved in these interactions was mapped and it was determined that the clade-specific CRF domain alone is sufficient for these interactions. This is the first described function for the CRF domain in plants.

Three Strains of Tobacco etch virus Distinctly Alter the Transcriptome of Apical Stem Tissue in Capsicum annuum during Infection.

Tobacco etch virus (TEV; genus Potyvirus) is flexuous rod shaped with a single molecule of single-stranded RNA and causes serious yield losses in species in the Solanaceae. Three TEV strains (HAT, Mex21, and N) are genetically distinct and cause different disease symptoms in plants. Here, a transcriptomic RNA sequencing approach was taken for each TEV strain to evaluate gene expression of the apical stem segment of pepper plants during two stages of disease development. Distinct profiles of Differentially Expressed Genes (DEGs) were identified for each TEV strain. DEG numbers increased with degree of symptom severity: 24 from HAT, 1190 from Mex21, and 4010 from N. At 7 days post-inoculation (dpi), when systemic symptoms were similar, there were few DEGs for HAT- and Mex21-infected plants, whereas N-infected plants had 2516 DEGs. DEG patterns from 7 to 14 dpi corresponded to severity of disease symptoms: milder disease with smaller DEG changes for HAT and Mex21 and severe disease with larger DEG changes for N. Strikingly, in each of these comparisons, there are very few overlapping DEGs among the TEV strains, including no overlapping DEGs between all three strains at 7 or 14 dpi.

The CRF domain defines cytokinin response factor proteins in plants.

BACKGROUND: Cytokinin Response Factors (CRFs) are a small subset of AP2/ERF transcription factor genes shown in Arabidopsis to regulate leaf development as part of the cytokinin signal transduction pathway. This study examines the phylogenetic distribution of CRF genes in other plant species, and attempts to identify the extent of sequence conservation and potential gene function among all CRF genes. RESULTS: We identified CRF genes in representatives of all major land plant lineages, including numerous flowering plant taxa in addition to the model systems in which ERF genes have been catalogued. Comparative analysis across this broader sampling has identified strongly conserved amino acid motifs other than the AP2/ERF domain for all CRF proteins as well as signature sequences unique to specific clades of CRF genes. One of these motifs, here designated as the CRF domain, is conserved in and unique to CRF proteins distinguishing them from related genes. We show that this novel domain of approximately 65 amino acids is found in CRF proteins from all groups of land plants and only in CRF genes. Phylogenetic analyses suggest that the evolution of CRF genes has included numerous duplication events. In this phylogenetic context we examine protein evolution including the gain and loss of accessory domains, correlate these molecular evolutionary events with experimental data on cytokinin regulation and speculate on the function and evolution of the CRF domain within AP2/ERF transcription factor proteins. We also tested a prediction drawn from the phylogenetic analyses that four CRF domain containing genes from Tomato, previously unexamined for cytokinin response, are transcriptionally inducible by cytokinin, supporting the link between CRF genes, CRF-specific domains and cytokinin regulation. CONCLUSION: CRF genes can be identified in all lineages of land plants, as a distinct subset of AP2/ERF proteins containing a specific and unique CRF domain. The CRF domain can be used to identify previously unclassified predicted genes or genes identified only as members of the AP2/ERF protein family. CRF domain presence and phylogenetic relatedness to known Arabidopsis CRF genes predicts gene function to some extent.