ERF Subfamily Research Articles

Expression Patterns of GmAP2/EREB-Like Transcription Factors Involved in Soybean Responses to Water Deficit

Soybean farming has faced several losses in productivity due to drought events in the last few decades. However, plants have molecular mechanisms to prevent and protect against water deficit injuries, and transcription factors play an important role in triggering different defense mechanisms. Understanding the expression patterns of transcription factors in response to water deficit and to environmental diurnal changes is very important for unveiling water deficit stress tolerance mechanisms. Here, we analyzed the expression patterns of ten APETALA2/Ethylene Responsive Element Binding-like (AP2/EREB-like) transcription factors in two soybean genotypes (BR16: drought-sensitive; and Embrapa 48: drought-tolerant). According to phylogenetic and domain analyses, these genes can be included in the DREB and ERF subfamilies. We also analyzed a GmDRIP-like gene that encodes a DREB negative regulator. We detected the up-regulation of 9 GmAP2/EREB-like genes and identified transcriptional differences that were dependent on the levels of the stress applied and the tissue type analyzed (the expression of the GmDREB1F-like gene, for example, was four times higher in roots than in leaves). The GmDRIP-like gene was not induced by water deficit in BR16 during the longest periods of stress, but was significantly induced in Embrapa 48; this suggests a possible genetic/molecular difference between the responses of these cultivars to water deficit stress. Additionally, RNAseq gene expression analysis over a 24-h time course indicates that the expression patterns of several GmDREB-like genes are subject to oscillation over the course of the day, indicating a possible circadian regulation.

Ethylene response factors and their role in plant defence.

Abstract The APETALA2/Ethylene response factor (AP2/ERF) family of transcription factors (TFs) plays crucial roles in the regulation of gene expression during plant development and in response to biotic and abiotic stress. Although AP2/ERFs have been implicated in a range of plant stress responses, this review focuses on ERFs in the context of the plant response to other organisms, primarily not only pathogenic but also symbiotic. The ERF subfamily is particularly important in the establishment and the tight regulation of plant defences through a balance of positive and negative transcriptional regulation. The expression of the ERFs is induced by pathogens with different lifestyles and they have been implicated in resistance to biotrophs, necrotrophs and hemibiotrophs, but members also play important roles in other plant-microbe interactions, for example, nodule formation in legumes. ERFs achieve this, in part, by acting as integrators in the crosstalk between signalling pathways mediated by ethylene (ET), jasmonic acid, salicylic acid and other plant hormones to modulate gene expression according to the stimuli sensed. This review collates recent findings on the regulation of this family of TFs, including transcriptional and post-translational regulation. Although DNA binding is typically conferred through the characteristic AP2 domain, the function of other domains including the EDLL and ERF-associated amphiphilic repression (EAR) domains in regulating gene expression is discussed. Finally, the potential for ERFs to be used to enhance resistance to pathogens in crops is considered.

AP2/ERF Transcription Factor in Rice: Genome-Wide Canvas and Syntenic Relationships between Monocots and Eudicots.

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.

Bioinformatic prediction of the AP2/ERF family genes in Eucalyptus grandis: focus on the CBF family

Background Due to their very high economic importance, Eucalyptus tree species are among the most planted hardwoods in the world with over 20 million hectares. However, as long-lived evergreen species, this genus is particularly exposed to cold. Frost tolerance varies among species and is inversely correlated to productivity. The AP2/ERF gene family includes developmentally and physiologically important transcription factors characterized by the presence of the AP2/ERF DNA-binding domain. AP2 proteins contain two AP2-like domains and RAV family proteins contain one AP2 domain and one B3 domain. ERF family proteins exhibit only one AP2 domain and are further divided into the DREB subfamily and the ERF subfamily [1]. The CBF/DREB1 protein differ from the other DREB proteins by the presence of “signature sequences” (PKK/RPAGRxKFxETRHP and DSAWR) flanking the DNA-binding AP2 domain [2]. The DREB factors recognize the C-repeat or dehydration response element (DRE) in the promoters of low temperature and/or water deficit responsive genes and would play a crucial role in response to abiotic stresses. CBF/DREB1 are the key regulators of the cold-responsive (COR) genes. So far CBF transcription factors have been mainly characterized in model plants such as Arabidopsis, but lately they were identified in several tree species including Eucalyptus [2]. The Eucalyptus cold tolerance was greatly improved in our hands when two genes from the four CBF members isolated from a tolerant species E. gunnii were individually constitutively overexpressed in the frost sensitive E. urophylla x E. grandis hybrid [3]. In the present study E. grandis AP2/ERF family genes were identified based on the presence of putative encoding AP2-domain(s) and were studied with regard to the model herbaceous Arabidopsis as well as the main sequenced woody plants. Within this family, a part of the study focused on the CBF/DREB1 subfamily which was compared to the four genes already characterized in E. gunnii[2].

Functions and Application of the AP2/ERF Transcription Factor Family in Crop ImprovementF

Plants have acquired sophisticated stress response systems to adapt to changing environments. It is important to understand plants' stress response mechanisms in the effort to improve crop productivity under stressful conditions. The AP2/ERF transcription factors are known to regulate diverse processes of plant development and stress responses. In this study, the molecular characteristics and biological functions of AP2/ERFs in a variety of plant species were analyzed. AP2/ERFs, especially those in DREB and ERF subfamilies, are ideal candidates for crop improvement because their overexpression enhances tolerances to drought, salt, freezing, as well as resistances to multiple diseases in the transgenic plants. The comprehensive analysis of physiological functions is useful in elucidating the biological roles of AP2/ERF family genes in gene interaction, pathway regulation, and defense response under stress environments, which should provide new opportunities for the crop tolerance engineering.

Expression profiles and transactivation analysis of a novel ethylene-responsive transcription factor gene GhERF5 from cotton

Plant AP2/EREBP transcription factors play important roles in plant development and in plant responses to biotic and abiotic stresses. A novel gene for ethylene-responsive element binding protein (EREBP), designated GhERF5, which encodes a protein of 255 amino acids, was isolated by RACE-PCR from cotton ( Gossypium hirsutum) seedlings. Sequence alignment revealed that GhERF5 contains a typical AP2/ERF domain, and belongs to the B3 subgroup of the ERF subfamily. Particle bombardment assay showed that GhERF5 functions as an in vivo transcription activator in tobacco cells, and it is located in the nuclei of onion epidermis cells. Semi-quantitative RT-PCR revealed that the expression of GhERF5 was highly and rapidly induced when plants were treated with exogenous ethylene, abscisic acid (ABA), salt, cold and drought. Promoter analysis indicated that there are conserved cis-acting elements induced by these stresses in the 5′-upstream region of the GhERF5 gene. These results suggest that the GhERF5 gene might play an important role in cotton response to ethylene, ABA and environmental stresses.

The transcription factor MtSERF1 may function as a nexus between stress and development in somatic embryogenesis in Medicago truncatula

In Medicago truncatula high rates of somatic embryo formation can be induced in the Jemalong genotype 2HA by application of the hormones auxin and cytokinin. Biosynthesis of the stress-related hormone ethylene is also necessary for somatic embryogenesis (SE) and is most likely a response to wounding and the presence of auxin in the medium. We have demonstrated that expression of a gene designated Mt SOMATIC EMBRYO RELATED FACTOR 1 (MtSERF1) induced by ethylene, in the presence of auxin plus cytokinin, is essential for SE. The promoter region of this transcription factor, a member of the ERF sub-family of the AP2/ERF super family, contains putative binding sites for auxin and cytokinin in addition to ethylene. An additional finding was the presence of WUSCHEL (WUS) binding sites in the MtSERF1 promoter region, which is discussed. Here we also discuss the Medicago data in the context of embryogenesis studies in Arabidopsis and suggest that MtSERF1 has a key developmental role, possibly in conjunction with WUS, in regulating downstream genes required for the initiation of SE.Addendum to: Mantiri FR, Kurdyukov S, Lohar DP, Sharopova N, Saeed NA, Wang X-D, VandenBosch KA, Rose RG. The transcription factor MtSERF1 of the ERF subfamily identified by transcriptional profiling is required for somatic embryogenesis induced by auxin plus cytokinin in Medicago truncatula. Plant Physiol 2008; 146:1622-36.

Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa

Populus is a model system for investigating the wood development, crown formation, and disease resistance in perennial plants. AR2/ERF is a large family of transcription factors in plant, encoding transcriptional regulators with a variety of functions involved in the developmental and physiological processes. Here, starting from database of Populus genome, we identified 200 AP2/ERF genes by in silico cloning method using the AP2/ERF conserved domain amino acid sequence of Arabidopsis thaliana as probe. Based on the number of AP2/ERF domains and the function of the genes, those AP2/ERF genes from Populus were classified into four subfamilies named the AP2, DREB, ERF, RAV, and a soloist. Among these genes, the number genes of total AP2/ERF family genes, DREB subfamily, and ERF subfamily from Populus trichocarpa were about 1.4–1.6-fold than those from A. thaliana. The rates were very similar for the putative homologs between Populus and Arabidopsis.

The Transcription Factor MtSERF1 of the ERF Subfamily Identified by Transcriptional Profiling Is Required for Somatic Embryogenesis Induced by Auxin Plus Cytokinin in Medicago truncatula

Transcriptional profiling of embryogenic callus produced from Medicago truncatula mesophyll protoplasts indicated up-regulation of ethylene biosynthesis and ethylene response genes. Using inhibitors of ethylene biosynthesis and perception, it was shown that ethylene was necessary for somatic embryogenesis (SE) in this model legume. We chose several genes involved in ethylene biosynthesis and response for subsequent molecular analyses. One of these genes is a gene encoding a transcription factor that belongs to the AP2/ERF superfamily and ERF subfamily of transcription factors. We demonstrate that this gene, designated M. truncatula SOMATIC EMBRYO RELATED FACTOR1 (MtSERF1), is induced by ethylene and is expressed in embryogenic calli. MtSERF1 is strongly expressed in the globular somatic embryo and there is high expression in a small group of cells in the developing shoot meristem of the heart-stage embryo. RNA interference knockdown of this gene causes strong inhibition of SE. We also provide evidence that MtSERF1 is expressed in zygotic embryos. MtSERF1 appears to be essential for SE and may enable a connection between stress and development.

Transcriptional Response to Nod Signaling

Nitrogen fixation occurs in the root nodules of legumes that form as part of a symbiotic process with rhizobial bacteria. Although the transcriptional regulators of the GRAS family were identified as important for mediating the plant response to the bacterial Nod factor that induces nodule formation, these GRAS proteins may not directly bind DNA. Middleton et al . identify ERN, a member of the ERF subfamily of transcription factors with the AP2 domain that bind DNA, as a participant in Nod signaling in Medicago truncatula . The authors screened plants deficient in nodule formation and identified two alleles called bit1-1 and bit1-2 . Plants carrying either of these alleles not only failed to form nodules in response to rhizobial infection but also failed to form nodules in response to a constitutively active form of calcium- and calmodulin-dependent protein kinase (CCaMK) that causes spontaneous nodule formation. The bit1-1 plants were also defective in activation of a Nod factor reporter gene. Genetic mapping of these alleles revealed that they encoded point mutations in the DNA binding domain ( bit1-2 ) or deletions ( bit1-1 ) in a gene that encodes a protein with sequence similarity to ERF transcription factors. They named this protein ERN for ERF Required for Nodulation. Rhizobial inoculation stimulated the expression of ERN , and this required a functional Nod signaling response. Consistent with a role in transcriptional response to Nod signaling, microarray analysis of plants with the bit1-1 allele showed decreased transcriptional responsiveness to rhizobial infection. Whether ERN interacts with GRAS transcriptional regulators and whether ERN binds to the promoters of Nod-regulated genes remains to be determined. P. H. Middleton, J. Jakab, R. V. Penmetsa, C. G. Starker, J. Doll, P. Kaló, R. Prabhu, J. F. Marsh, R. M. Mitra, A. Kereszt, B. Dudas, K. VandenBosch, S. R. Long, D. R. Cook, G. B. Kiss, G. E. D. Oldroyd, An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction. Plant Cell 19 , 1221-1234 (2007). [Abstract] [Full Text]

Two novel AP2/ERF domain proteins interact with cis‐element VWRE for wound‐induced expression of the Tobacco tpoxN1 gene

The vascular system-specific and wound-responsive cis-element (VWRE) has been identified as a novel cis-element for wound-induced and vascular system-specific expression of the tobacco peroxidase gene, tpoxN1. Here we isolated two independent clones that encode VWRE binding proteins by yeast one-hybrid screening. As the gene products have an AP2/ERF (APETALA2/ethylene-responsive factor) domain, and the transcripts were accumulated transiently after wounding, we named them wound-responsive AP2/ERF-like factor 1 (WRAF1) and WRAF2. The AP2/ERF domains of the two WRAFs share 97% homology, and are classified into the ERF subfamily B-4. Gel mobility shift analysis indicated that WRAFs specifically bind VWRE, which contains no known cis-elements for other AP2/ERF proteins. The binding activity of the WRAFs was found to be localized in the AP2/ERF domain. The WRAFs transactivated a promoter containing four tandem repeats of the VWRE, but not that of the mutated VWRE. Overexpression of the WRAF genes led to constitutive expression of the potential target gene, tpoxN1, in unwounded transgenic plants. These results indicate that the novel transcription factors WRAF1 and WRAF2 bind the VWRE as positive regulators for the expression of the tpoxN1 gene.

Isolation and functional characterization of the JcERF gene, a putative AP2/EREBP domain-containing transcription factor, in the woody oil plant Jatropha curcas

A cDNA clone, named JcERF, was isolated from Jatropha curcas seedlings (a woody oil plant). It was classified as an ERF subfamily member based on multiple sequence alignment and phylogenetic characterization. The deduced amino acid sequences of the JcERF clone showed no significant sequence similarity with other known ERF proteins except for the conserved AP2/EREBP DNA-binding domain. Expression of the JcERF gene was rapidly induced upon salinity, drought, ethylene and mechanical wounding treatments. No significant changes in the JcERF expression were observed under ABA stress. Gel retardation assay revealed that the JcERF protein could bind specifically to the GCC box as well as to the C/DRE motif. Also it can be inferred from the gel-shift that there is a possibility that the near sequence of the GCC box has an important effect on the DNA-binding activity. In yeast, the JcERF protein specifically bound to the DRE sequence and activated the transcription of two reporter genes His3 and LacZ driven by the DRE sequence. When fused to the LexA DNA-binding domain, the full-length JcERF functioned effectively as a trans-activator in the yeast one-hybrid assay. Overexpression of JcERF cDNA in transgenic Arabidopsis enhanced the salt and freezing tolerance. Meanwhile the seed germination of JcERF transgenic plants was not affected by various concentrations ABA in MS medium. Taken together, the results showed that JcERF functioned as a novel transcription factor and it exhibited a mechanism of plant response to environmental factors like the other AP2/EREBP regulons that also exist in tropical woody plants.

DNA-Binding Specificity of the ERF/AP2 Domain of Arabidopsis DREBs, Transcription Factors Involved in Dehydration- and Cold-Inducible Gene Expression

DRE/CRT is a cis-acting element that is involved in gene expression responsive to drought and low-temperature stress in higher plants. DREB1A/CBF3 and DREB2A are transcription factors that specifically bind to DRE/CRT in Arabidopsis. We precisely analyzed the DNA-binding specificity of DREBs. Both DREBs specifically bound to six nucleotides (A/GCCGAC) of DRE. However, these proteins had different binding specificities to the second or third nucleotides of DRE. Gel mobility shift assay using mutant DREB proteins showed that the two amino acids, valine and glutamic acid conserved in the ERF/AP2 domains, especially valine, have important roles in DNA-binding specificity. In the Arabidopsis genome, 145 DREB/ERF-related proteins are encoded. These proteins were classified into five groups—AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others. The DREB subfamily included three novel DREB1A- and six DREB2A-related proteins. We analyzed expression of novel genes for these proteins and discuss their roles in stress-responsive gene expression.