The CRF domain defines cytokinin response factor proteins in plants.
ABSTRACT: 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.
Project description:The AP2/ERF transcription factor family is one of the largest families involved in growth and development, hormone responses, and biotic or abiotic stress responses in plants. In this study, 281 AP2/ERF transcription factor unigenes were identified in Chinese cabbage. These superfamily members were classified into three families (AP2, ERF, and RAV). The ERF family was subdivided into the DREB subfamily and the ERF subfamily with 13 groups (I- XI) based on sequence similarity. Duplication, evolution and divergence of the AP2/ERF genes in B. rapa and Arabidopsis thaliana were investigated and estimated. Cytokinin response factors (CRFs), as a subclade of the AP2/ERF family, are important transcription factors that define a branch point in the cytokinin two-component signal (TCS) transduction pathway. Up to 21 CRFs with a conserved CRF domain were retrieved and designated as BrCRFs. The amino acid sequences, conserved regions and motifs, phylogenetic relationships, and promoter regions of the 21 BrCRFs were analyzed in detail. The BrCRFs broadly expressed in various tissues and organs. The transcripts of BrCRFs were regulated by factors such as drought, high salinity, and exogenous 6-BA, NAA, and ABA, suggesting their involvement in abiotic stress conditions and regulatory mechanisms of plant hormone homeostasis. These results provide new insight into the divergence, variation, and evolution of AP2/ERF genes at the genome-level in Chinese cabbage.
Project description:Cytokinin Response Factor (CRF) genes are a subgroup of AP2/ERF domain-containing transcription factors that are defined by the CRF domain, from which five clades of CRF genes have been identified. Clade III CRFs are strongly induced by cytokinin, as well as other abiotic stress factors, such as oxidative stress. While this appears well studied for the Clade III CRFs in Arabidopsis and tomato, there have been almost no studies done outside of these model systems. This study expands upon that and represents the first CRF research in the Sunflower family, Asteraceae. Fifty Asterid Clade III CRF protein sequences were examined, and novel Clade III CRF C-terminus motifs were identified. Clade III CRF genes of Marshallia mohrii and M. caespitosa were assembled from genome-skimming and transcriptomic data. Expression experiments were conducted on M. caespitosa to test responsiveness to both cytokinin and oxidative stress. Low levels of basal expression for the McCRF1 were found to be strongly induced in both treatment groups. These are the first experiments to show regulation of a nuclear gene in a Marshallia species, and these results suggest there is broad conservation in the sequence, form, and regulation of Clade III CRF genes and proteins.
Project description: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.
Project description:The transcription factor family intimately regulates gene expression in response to hormones, biotic and abiotic factors, symbiotic interactions, cell differentiation, and stress signalling pathways in plants. In this study, 170 AP2/ERF family genes are identified by phylogenetic analysis of the rice genome (Oryza sativa l. japonica) and they are divided into a total of 11 groups, including four major groups (AP2, ERF, DREB, and RAV), 10 subgroups, and two soloists. Gene structure analysis revealed that, at position-6, the amino acid threonine (Thr-6) is conserved in the double domain AP2 proteins compared to the amino acid arginine (Arg-6), which is preserved in the single domain of ERF proteins. In addition, the histidine (His) amino acid is found in both domains of the double domain AP2 protein, which is missing in single domain ERF proteins. Motif analysis indicates that most of the conserved motifs, apart from the AP2/ERF domain, are exclusively distributed among the specific clades in the phylogenetic tree and regulate plausible functions. Expression analysis reveals a widespread distribution of the rice AP2/ERF family genes within plant tissues. In the vegetative organs, the transcripts of these genes are found most abundant in the roots followed by the leaf and stem; whereas, in reproductive tissues, the gene expression of this family is observed high in the embryo and lemma. From chromosomal localization, it appears that repetition and tandem-duplication may contribute to the evolution of new genes in the rice genome. In this study, interspecies comparisons between rice and wheat reveal 34 rice loci and unveil the extent of collinearity between the two genomes. It was subsequently ascertained that chromosome-9 has more orthologous loci for CRT/DRE genes whereas chromosome-2 exhibits orthologs for ERF subfamily members. Maximum conserved synteny is found in chromosome-3 for AP2 double domain subfamily genes. Macrosynteny between rice and Arabidopsis, a distant, related genome, uncovered 11 homologs/orthologs loci in both genomes. The distribution of AP2/ERF family gene paralogs in Arabidopsis was most frequent in chromosome-1 followed by chromosome-5. In Arabidopsis, ERF subfamily gene orthologs are found on chromosome-1, chromosome-3, and chromosome-5, whereas DRE subfamily genes are found on chromosome-2 and chromosome-5. Orthologs for RAV and AP2 with double domains in Arabidopsis are located on chromosome-1 and chromosome-3, respectively. In conclusion, the data generated in this survey will be useful for conducting genomic research to determine the precise role of the AP2/ERF gene during stress responses with the ultimate goal of improving crops.
Project description:Apetala2-ethylene-responsive element binding factor (AP2-ERF) superfamily with common AP2-DNA binding domain have developmentally and physiologically important roles in plants. Since common bean genome project has been completed recently, it is possible to identify all of the AP2-ERF genes in the common bean genome. In this study, a comprehensive genome-wide in silico analysis identified 180 AP2-ERF superfamily genes in common bean (Phaseolus vulgaris). Based on the amino acid alignment and phylogenetic analyses, superfamily members were classified into four subfamilies: DREB (54), ERF (95), AP2 (27) and RAV (3), as well as one soloist. The physical and chemical characteristics of amino acids, interaction between AP2-ERF proteins, cis elements of promoter region of AP2-ERF genes and phylogenetic trees were predicted and analyzed. Additionally, expression levels of AP2-ERF genes were evaluated by in silico and qRT-PCR analyses. In silico micro-RNA target transcript analyses identified nearly all PvAP2-ERF genes as targets of by 44 different plant species' miRNAs were identified in this study. The most abundant target genes were PvAP2/ERF-20-25-62-78-113-173. miR156, miR172 and miR838 were the most important miRNAs found in targeting and BLAST analyses. Interactome analysis revealed that the transcription factor PvAP2-ERF78, an ortholog of Arabidopsis At2G28550, was potentially interacted with at least 15 proteins, indicating that it was very important in transcriptional regulation. Here we present the first study to identify and characterize the AP2-ERF transcription factors in common bean using whole-genome analysis, and the findings may serve as a references for future functional research on the transcription factors in common bean.
Project description:The AP2/ERF superfamily, one of the most important transcription factor families, plays crucial roles in response to biotic and abiotic stresses. So far, a comprehensive evolutionary inference of its origination and expansion has not been available. Here, we identified 515 AP2/ERF genes in B. napus, a neo-tetraploid forming ~7500 years ago, and found that 82.14% of them were duplicated in the tetraploidization. A prominent subgenome bias was revealed in gene expression, tissue-specific, and gene conversion. Moreover, a large-scale analysis across plants and alga suggested that this superfamily could have been originated from AP2 family, expanding to form other families (ERF, and RAV). This process was accompanied by duplicating and/or alternative deleting AP2 domain, intragenic domain sequence conversion, and/or by acquiring other domains, resulting in copy number variations, alternatively contributing to functional innovation. We found that significant positive selection occurred at certain critical nodes during the evolution of land plants, possibly responding to changing environment. In conclusion, the present research revealed origination, functional innovation, and evolutionary trajectory of the AP2/ERF superfamily, contributing to understanding their roles in plant stress tolerance.
Project description:BACKGROUND: Chinese cabbage (Brassica rapa ssp. pekinensis) is a member of one of the most important leaf vegetables grown worldwide, which has experienced thousands of years in cultivation and artificial selection. The entire Chinese cabbage genome sequence, and more than forty thousand proteins have been obtained to date. The genome has undergone triplication events since its divergence from Arabidopsis thaliana (13 to 17 Mya), however a high degree of sequence similarity and conserved genome structure remain between the two species. Arabidopsis is therefore a viable reference species for comparative genomics studies. Variation in the number of members in gene families due to genome triplication may contribute to the broad range of phenotypic plasticity, and increased tolerance to environmental extremes observed in Brassica species. Transcription factors are important regulators involved in plant developmental and physiological processes. The AP2/ERF proteins, one of the most important families of transcriptional regulators, play a crucial role in plant growth, and in response to biotic and abiotic stressors. Our analysis will provide resources for understanding the tolerance mechanisms in Brassica rapa ssp. pekinensis. RESULTS: In the present study, 291 putative AP2/ERF transcription factor proteins were identified from the Chinese cabbage genome database, and compared with proteins from 15 additional species. The Chinese cabbage AP2/ERF superfamily was classified into four families, including AP2, ERF, RAV, and Soloist. The ERF family was further divided into DREB and ERF subfamilies. The AP2/ERF superfamily was subsequently divided into 15 groups. The identification, classification, phylogenetic reconstruction, conserved motifs, chromosome distribution, functional annotation, expression patterns, and interaction networks of the AP2/ERF transcription factor superfamily were predicted and analyzed. Distribution mapping results showed AP2/ERF superfamily genes were localized on the 10 Chinese cabbage chromosomes. AP2/ERF transcription factor expression levels exhibited differences among six tissue types based on expressed sequence tags (ESTs). In the AP2/ERF superfamily, 214 orthologous genes were identified between Chinese cabbage and Arabidopsis. Orthologous gene interaction networks were constructed, and included seven CBF and four AP2 genes, primarily involved in cold regulatory pathways and ovule development, respectively. CONCLUSIONS: The evolution of the AP2/ERF transcription factor superfamily in Chinese cabbage resulted from genome triplication and tandem duplications. A comprehensive analysis of the physiological functions and biological roles of AP2/ERF superfamily genes in Chinese cabbage is required to fully elucidate AP2/ERF, which provides us with rich resources and opportunities to understand crop stress tolerance mechanisms.
Project description:The plant hormone cytokinin regulates numerous growth and developmental processes. A signal transduction pathway for cytokinin has been elucidated that is similar to bacterial two-component phosphorelays. In Arabidopsis, this pathway is comprised of receptors that are similar to sensor histidine kinases, histidine-containing phosphotransfer proteins, and response regulators (ARRs). There are two classes of response regulators, the type-A ARRs, which act as negative regulators of cytokinin responses, and the type-B ARRs, which are transcription factors that play a positive role in mediating cytokinin-regulated gene expression. Here we show that several closely related members of the Arabidopsis AP2 gene family of unknown function are transcriptionally up-regulated by cytokinin through this pathway, and we have designated these AP2 genes CYTOKININ RESPONSE FACTORS (CRFs). In addition to their transcriptional regulation by cytokinin, the CRF proteins rapidly accumulate in the nucleus in response to cytokinin, and this relocalization depends on the histidine kinases and the downstream histidine-containing phosphotransfer proteins, but is independent of the ARRs. Analysis of loss-of-function mutations reveals that the CRFs function redundantly to regulate the development of embryos, cotyledons, and leaves. Furthermore, the CRFs mediate a large fraction of the transcriptional response to cytokinin, affecting a set of cytokinin-responsive genes that largely overlaps with type-B ARR targets. These results indicate that the CRF proteins function in tandem with the type-B ARRs to mediate the initial cytokinin response. Thus, the evolutionarily ancient two-component system that is used by cytokinin branches to incorporate a unique family of plant-specific transcription factors.
Project description:AP2/ERF genes encode transcriptional regulators with a variety of functions in plant growth and development and in response to biotic and abiotic stresses. To date, there are no detailed classification and expression profiles for AP2/ERF genes in Salix. In this study, a comprehensive computational analysis identified 173 AP2/ERF superfamily genes in willow (Salix arbutifolia), by using in silico cloning methods with the use of the AP2/ERF conserved domain amino acid sequence of Arabidopsis thaliana as a probe. Based on the results of phylogenetic analyses and the number of AP2/ERF domains, the AP2/ERF genes were classified into four groups: AP2, RAV, ERF and Soloist. The expression profile was analyzed using transcriptome data from different tissues. A comparative analysis of AP2/ERF superfamily genes among Salix, Populus and Arabidopsis was performed. The Salix DREB, AP2 and RAV groups had a similar number to those in Arabidopsis, and the size of the ERF subfamily in Salix was about 1.4-fold that of Arabidopsis. The Salix DREB subfamily was smaller compared to Populus, while the other families were similar in size to those in Populus. These results will be useful for future functional analyses of the ERF family genes.
Project description:The APETALA2/ethylene-responsive element binding factor (AP2/ERF) family is one of the largest transcription factor (TF) families in plants that includes four major sub-families, namely AP2, DREB (dehydration responsive element binding), ERF (ethylene responsive factors) and RAV (Related to ABI3/VP). AP2/ERFs are known to play significant roles in various plant processes including growth and development and biotic and abiotic stress responses. Considering this, a comprehensive genome-wide study was conducted in foxtail millet (Setaria italica L.). A total of 171 AP2/ERF genes were identified by systematic sequence analysis and were physically mapped onto nine chromosomes. Phylogenetic analysis grouped AP2/ERF genes into six classes (I to VI). Duplication analysis revealed that 12 (?7%) SiAP2/ERF genes were tandem repeated and 22 (?13%) were segmentally duplicated. Comparative physical mapping between foxtail millet AP2/ERF genes and its orthologs of sorghum (18 genes), maize (14 genes), rice (9 genes) and Brachypodium (6 genes) showed the evolutionary insights of AP2/ERF gene family and also the decrease in orthology with increase in phylogenetic distance. The evolutionary significance in terms of gene-duplication and divergence was analyzed by estimating synonymous and non-synonymous substitution rates. Expression profiling of candidate AP2/ERF genes against drought, salt and phytohormones revealed insights into their precise and/or overlapping expression patterns which could be responsible for their functional divergence in foxtail millet. The study showed that the genes SiAP2/ERF-069, SiAP2/ERF-103 and SiAP2/ERF-120 may be considered as potential candidate genes for further functional validation as well for utilization in crop improvement programs for stress resistance since these genes were up-regulated under drought and salinity stresses in ABA dependent manner. Altogether the present study provides new insights into evolution, divergence and systematic functional analysis of AP2/ERF gene family at genome level in foxtail millet which may be utilized for improving stress adaptation and tolerance in millets, cereals and bioenergy grasses.