ERF-associated Amphiphilic Repression Research Articles

Overexpression of a Chinese cabbage BrERF11 transcription factor enhances disease resistance to Ralstonia solanacearum in tobacco

Ethylene-responsive factors (ERFs) play diverse roles in plant growth, developmental processes and stress responses. However, the roles and underlying mechanism of ERFs remain poorly understood, especially in non-model plants. In this study, a full length cDNA of ERF gene was isolated from the cDNA library of Chinese cabbage. According to sequence alignment, we found a highly conservative AP2/ERF domain, two nuclear localization signals, and an ERF-associated Amphiphilic Repression (EAR) motif in its C-terminal region. It belonged to VIIIa group ERFs sharing the highest sequence identity with AtERF11 in all of the ERFs in Arabidopsis and designated BrERF11. BrERF11-green fluorescence protein (GFP) transient expressed in onion epidermis cells localized to the nucleus. The transcript levels of BrERF11 were induced by exogenous salicylic acid (SA), methyl jasmonate (MeJA), ethephon (ETH), and hydrogen peroxide (H(2)O(2)). Constitutive expression of BrERF11 enhanced tolerance to Ralstonia solanacearum infection in transgenic tobacco plants, which was coupled with hypersensitive response (HR), burst of H(2)O(2) and upregulation of defense-related genes including HR marker genes, SA-, JA-dependent pathogen-related genes and ET biosynthesis associated genes and downregulation of CAT1, suggesting BrERF11 may participate in pathogen-associated molecular pattern (PAMP)- and effector-triggered immunity (PTI and ETI) mediated by SA-, JA- and ET-dependent signaling mechanisms.

Over-expression of a subgroup 4 R2R3 type MYB transcription factor gene from Leucaena leucocephala reduces lignin content in transgenic tobacco

KEY MESSAGE : LlMYB1 , a subgroup 4 R2R3-type MYB transcription factor gene from Leucaena leucocephala appears to be a repressor of lignin biosynthesis pathway by regulating the transcription of general phenylpropanoid pathway genes. R2R3MYB transcription factors are known to play a wide role in regulating the phenylpropanoid pathway in plants. In this study, we report isolation, cloning and characterization of an R2R3MYB transcription factor gene (LlMYB1) from an economically important tree species, Leucaena leucocephala. LlMYB1 consists of 705bp coding sequence corresponding to 235 amino acids. Sequence alignment revealed that the N-terminal (MYB) domain of the gene shares up to 95% similarity with subgroup 4 (Sg4) members of R2R3Myb gene family functionally known to be lignin repressors. Highly divergent C-terminal region of the gene carried an ERF-associated amphiphilic repression (EAR) motif, another characteristic of the Sg4. The gene was phylogenetically grouped closest with AmMYB308, a known repressor of monolignol biosynthetic pathway genes. Spatio-temporal expression studies at different ages of seedlings using quantitative real-time PCR (QRT-PCR) showed highest transcript level of the gene in 10day old stem tissues. Over-expression of the gene in transgenic tobacco showed statistically significant decline in the transcript levels of the general phenylpropanoid pathway genes and reduction in lignin content. Our study suggests that LlMYB1 might be playing the role of a repressor of lignin biosynthesis in L. leucocephala.

Isolation and characterization of a bread wheat salinity responsive ERF transcription factor

A screen conducted on both a suppression subtractive hybridization and a full length cDNA library made from a salinity tolerant bread wheat cultivar SR3 (Triticum aestivum cv. SR3) resulted in the recognition of TaERF4, a gene including both an AP2/ERF domain and a nuclear localization signal. The 982bp TaERF4 cDNA comprised a 582bp open reading frame, encoding a 193 residue polypeptide of molecular weight 20kDa and calculated pI 8.48. A TaERF4-GFP fusion protein localized preferentially to the nuclei of Arabidopsis thaliana protoplasts. TaERF4 is a member of the B-1 group within the ERF sub‐family and was not transactivatable in yeast. The presence of an ERF-associated amphiphilic repression (EAR) motif at its C-terminus suggests that TaERF4 is probably a transcription repressor. TaERF4 was inducible by exposure to salinity and osmotic stresses, but not to exogenously supplied abscisic acid (ABA). The heterologous constitutive expression of TaERF4 in Arabidopsis enhanced the level of sensitivity to salinity stress, possibly via the repression of tonoplast Na+/H+ antiporter activity. There was no phenotype associated with the transgene's presence when plants were subjected to either osmotic stress or ABA treatment. TaERF4 appears to be a transcription repressor acting within the ABA-independent response to salinity stress.

Isolation and characterization of a novel transcriptional repressor GmERF6 from soybean

A new ethylene response factor (ERF), GmERF6, was isolated from soybean. Protein sequence alignment of GmERF6 revealed an AP2/ERF domain, two putative nuclear localization signals (NLSs) and an ERF-associated amphiphilic repression (EAR) motif. Real-time quantitative PCR analysis revealed that the expression of GmERF6 was differentially induced in soybean seedlings by drought, salt, cold, salicylic acid, ethylene, abscisic acid and methyl jasmonate. Transient expression experiments demonstrated that GmERF6 functions as a transcriptional repressor to downregulate the transcriptional levels of the reporter gene and repress the activated ability of other transcriptional activator. Transgenic Arabidopsis lines constitutively expressing GmERF6 showed an increased tolerance to drought compared to wild-type plants.

The EDLL motif: a potent plant transcriptional activation domain from AP2/ERF transcription factors

In plants, the ERF/EREBP family of transcriptional regulators plays a key role in adaptation to various biotic and abiotic stresses. These proteins contain a conserved AP2 DNA-binding domain and several uncharacterized motifs. Here, we describe a short motif, termed 'EDLL', that is present in AtERF98/TDR1 and other clade members from the same AP2 sub-family. We show that the EDLL motif, which has a unique arrangement of acidic amino acids and hydrophobic leucines, functions as a strong activation domain. The motif is transferable to other proteins, and is active at both proximal and distal positions of target promoters. As such, the EDLL motif is able to partly overcome the repression conferred by the AtHB2 transcription factor, which contains an ERF-associated amphiphilic repression (EAR) motif. We further examined the activation potential of EDLL by analysis of the regulation of flowering time by NF-Y (nuclear factor Y) proteins. Genetic evidence indicates that NF-Y protein complexes potentiate the action of CONSTANS in regulation of flowering in Arabidopsis; we show that the transcriptional activation function of CONSTANS can be substituted by direct fusion of the EDLL activation motif to NF-YB subunits. The EDLL motif represents a potent plant activation domain that can be used as a tool to confer transcriptional activation potential to heterologous DNA-binding proteins.

JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors.

Overexpression of tobacco ethylene response factor NtERF3 gene and its homologues from tobacco and rice induces hypersensitive response-like cell death in tobacco

Among plant-specific transcription factors, ethylene response factors (ERFs) comprise one of the largest families. ERFs are unique to the plant kingdom and are considered to have crucial roles in plant response to various biotic and abiotic environmental stresses. Here, we report on the functional analysis of a transcriptional repressor, NtERF3, with regard to cell death associated with a hypersensitive response (HR), a plant-specific resistance reaction against pathogens. Expression of NtERF3 was upregulated during HR induction by Tobacco mosaic virus (TMV) infection in tobacco plants harboring the resistance N gene to TMV. Transient overexpression of NtERF3 by Agrobacterium-mediated gene delivery induced HR-like cell death in tobacco, associated with the production of reactive oxygen species and ion leakage. Deletion of the ERF-associated amphiphilic repression (EAR) motif from NtERF3 resulted in no induction of cell death, while the deletion had no effect on nuclear localization of the proteins. After virus-mediated gene delivery, similar results also were observed in tobacco without the N gene. In addition to NtERF3, other EAR motif-containing ERFs from tobacco, Nicotiana benthamiana and rice also induced cell death when overproduced in tobacco plants. The results suggested that many ERF genes encoding EAR motif-containing proteins might have the ability to induce cell death when overexpressed.

MYB80, a regulator of tapetal and pollen development, is functionally conserved in crops

The Arabidopsis AtMYB80 transcription factor (formerly AtMYB103) regulate genes essential for tapetal and pollen development. One of these genes, coding for an aspartic protease (UNDEAD), may control the timing of tapetal programmed cell death (PCD). In crop plants such as rice and wheat, abiotic stresses lead to abnormal tapetal development resulting in delayed PCD. Manipulation of AtMYB80 function has been used to develop a reversible male sterility system applicable to hybrid crop production. MYB80 homologs were cloned from wheat, rice, canola and cotton. The promoters of the homologs drove temporal and spatial expression patterns of the GUS reporter gene in the tapetum and microspores of Arabidopsis anthers identical to the AtMYB80 promoter. A short region is conserved in all five MYB80 promoters. The MYB80 homolog genes, driven by the AtMYB80 or their respective promoters, rescued the atmyb80 mutant, completely restoring male fertility. The canola MYB80 was fused to the EAR (ERF-associated amphiphilic repression) repressor and canola plants transgenic for the construct exhibited premature tapetal degradation and subsequent pollen abortion. The five MYB80 homologs all shared a 44 amino acid sequence immediately adjacent to the R2R3 domain which appears to be necessary for MYB80 function.

Stress-Responsive Mitogen-Activated Protein Kinases Interact with the EAR Motif of a Poplar Zinc Finger Protein and Mediate Its Degradation through the 26S Proteasome

Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

Isolation and identification of an AP2/ERF factor that binds an allelic cis-element of rice gene LRK6

Allelic expression of the rice yield-related gene, leucine-rich receptor-like kinase 6 (LRK6), in the hybrid of 93-11 (Oryza sativa L. subsp. Indica var. 93-11) and Nipponbare (O. sativa L. subsp. Japonica var. Nipponbare) is determined by allelic promoter cis-elements. Using deletion analysis of the LRK6 promoter, we identified two distinct regions that might contribute to LRK6 expression. Sequence alignment revealed differences in these LRK6 promoter regions in 93-11 and Nipponbare. One of the segments, named differential sequence of LRK6 promoter 2 (DSLP2), contains potential transcription factor binding sites. Using a yeast one-hybrid assay, we isolated an ethylene-responsive factor (ERF) protein that binds to DSLP2. Sequence analysis and a GCC-box assay showed that the ERF gene, O. sativa ERF 3 (OsERF3), which belongs to ERF subfamily class II, has a conserved ERF domain and an ERF-associated amphiphilic repression repressor motif. We used an in vivo mutation assay to identify a new motif (5'-TAA(A)GT-3') located in DSLP2, which interacts with OsERF3. These results suggest that OsERF3, an AP2 (APETALA 2 Gene)/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid.

Morphological changes of Rosa×hybrida by a chimeric repressor of Arabidopsis TCP3

Chimeric repressor gene-silencing technology is a useful tool for changing morphology of ornamental plants. It has previously been demonstrated that the chimeric repressor TCP3SRDX, which consists of Arabidopsis TCP3 and an ERF-associated amphiphilic repression motif repression domain, perturbs the marginal morphology of Arabidopsis leaves and flowers. To obtain new rose cultivars that have ornamental values, we attempted to alter the morphology of Rosa×hybrida cv. Lavande with TCP3SRDX. The TCP3SRDX transgenic rose plants showed interesting phenotypes: the number of leaflets and the size of leaf teeth increased, the petals were wavy, and the sepals were compound-leafy. We succeeded in altering rose morphology using Arabidopsis TCP3 without the sequence information of a TCP3 homologue in the target plant species.

Genome‐Wide Analysis of WOX Gene Family in Rice, Sorghum, Maize, Arabidopsis and Poplar

WUSCHEL-related homeobox (WOX) genes form a large gene family specifically expressed in plants. They are known to play important roles in regulating the development of plant tissues and organs by determining cell fate. Recent available whole genome sequences allow us to do more comprehensive phylogenetic analysis of the WOX genes in plants. In the present study, we identified 11 and 21 WOXs from sorghum (Sorghum bicolor) and maize (Zea mays), respectively. The 72 WOX genes from rice (Oryza sativa), sorghum, maize, Arabidopsis (Arabidopsis thaliana) and poplar (Populus trichocarpa) were grouped into three well supported clades with nine subgroups according to the amino acid sequences of their homodomains. Their phylogenetic relationship was also supported by the observation of the motifs outside the homodomain. We observed the variation of duplication events among the nine sub-groups between monocots and eudicots, for instance, more gene duplication events of WOXs within subgroup A for monocots, while, less for dicots in this subgroup. Furthermore, we observed the conserved intron/exon structural patterns of WOX genes in rice, sorghum and Arabidopsis. In addition, WUS (Wuschel)-box and EAR (the ERF-associated amphiphilic repression)-like motif were observed to be conserved among several WOX subgroups in these five plants. Comparative analysis of expression patterns of WOX genes in rice and Arabidopsis suggest that the WOX genes play conserved and various roles in plants. This work provides insights into the evolution of the WOX gene family and is useful for future research.

Ectopic expression of an EAR motif deletion mutant of SlERF3 enhances tolerance to salt stress and Ralstonia solanacearum in tomato

Ethylene-responsive transcription factors (ERFs) bind specifically to cis-acting DNA regulatory elements such as GCC boxes and play an important role in the regulation of defense- and stress-related genes in plants. In contrast to other ERFs, class II ERFs contain an ERF-associated amphiphilic repression (EAR) domain and act as GCC-mediated transcriptional repressors. In this study, SlERF3, a class II ERF was isolated from tomato and characterized. To examine whether the EAR motif of class II ERF proteins participates in ERF-mediated functions in plants, the EAR domain was deleted to generate SlERF3ΔRD. We show that SlERF3ΔRD protein retains the character of a transcription factor and becomes a GCC-mediated transcriptional activator. Constitutive expression of full-length SlERF3 in tomato severely suppressed growth and, as a result, no transgenic plants were obtained. However, no apparent effects on growth and development of SlERF3ΔRD transgenic plants were observed. Overexpression of SlERF3ΔRD in transgenic tomato induced expression of pathogenesis-related protein genes such as PR1, PR2 and PR5, and enhanced tolerance to Ralstonia solanacearum. Furthermore, transgenic Arabidopsis and tomatoes constitutively expressing SlERF3ΔRD exhibited reduced levels of membrane lipid peroxidation and enhanced tolerance to salt stress. In comparison with wild-type plants grown under stress conditions, transgenic SlERF3ΔRD tomatoes produced more flowers, fruits, and seeds. This study illustrates a gene-enhancing tolerance to both biotic and abiotic stresses in transgenic plants with the deletion of a repressor domain. Our findings suggest that class II ERF proteins may find important use in crop improvement or genetic engineering to increase stress tolerance in plants.

The cotton dehydration-responsive element binding protein GhDBP1 contains an EAR-motif and is involved in the defense response of Arabidopsis to salinity stress

Our previous work reported that cotton dehydration-responsive element (DRE) binding protein 1 (GhDBP1) could function as an active transcriptional repressor for DRE-mediated gene expression. However, the repression mechanism utilised by GhDBP1 was unclear. In this report, we demonstrate that GhDBP1’s transcriptional repression domain is located at the C-terminus, and is known as an ERF-associated amphiphilic repression (EAR)-motif. Furthermore, the amino acid residues aspartic acid (D), leucine (L), asparagine (N) and proline (P) are conserved in the EAR-motif, and were found to be necessary for repression through mutational analysis. In addition, our promoter assays demonstrated a dehydration-induced and rehydration-repressed expression pattern of GhDBP1. Transgenic Arabidopsis plants overexpressing GhDBP1 were more sensitive to high salinity stress and appeared to downregulate the expression levels of the stress-induced effecter genes. Taken together, our findings provide an important insight into GhDBP1’s potential molecular repression mechanism and how it is involved in plant stress responses.

The Arabidopsis EAR-motif-containing protein RAP2.1 functions as an active transcriptional repressor to keep stress responses under tight control

BackgroundPlants respond to abiotic stress through complex regulation of transcription, including both transcriptional activation and repression. Dehydration-responsive-element binding protein (DREB)-type transcription factors are well known to play important roles in adaptation to abiotic stress. The mechanisms by which DREB-type transcription factors activate stress-induced gene expression have been relatively well studied. However, little is known about how DREB-type transcriptional repressors modulate plant stress responses. In this study, we report the functional analysis of RAP2.1, a DREB-type transcriptional repressor.ResultsRAP2.1 possesses an APETALA2 (AP2) domain that binds to dehydration-responsive elements (DREs) and an ERF-associated amphiphilic repression (EAR) motif, as the repression domain located at the C-terminus of the protein. Expression of RAP2.1 is strongly induced by drought and cold stress via an ABA-independent pathway. Arabidopsis plants overexpressing RAP2.1 show enhanced sensitivity to cold and drought stresses, while rap2.1-1 and rap2.1-2 T-DNA insertion alleles result in reduced sensitivity to these stresses. The reduced stress sensitivity of the plant containing the rap2.1 allele can be genetically complemented by the expression of RAP2.1, but not by the expression of EAR-motif-mutated RAP2.1. Furthermore, chromatin immunoprecipitation (ChIP) analysis has identified Responsive to desiccation/Cold-regulated (RD/COR) genes as downstream targets of RAP2.1 in vivo. Stress-induced expression of the RD/COR genes is repressed by overexpression of RAP2.1 and is increased in plants expressing the rap2.1 allele. In addition, RAP2.1 can negatively regulate its own expression by binding to DREs present in its own promoter. Our data suggest that RAP2.1 acts as a negative transcriptional regulator in defence responses to cold and drought stress in Arabidopsis.ConclusionsA hypothetical model for the role of RAP2.1 in modulating plant responses to cold and drought is proposed in this study. It appears that RAP2.1 acts as a negative "subregulon" of DREB-type activators and is involved in the precise regulation of expression of stress-related genes, acting to keep stress responses under tight control.

Detection of protein-protein interactions in plants using the transrepressive activity of the EAR motif repression domain

The activities of many regulatory factors involve interactions with other proteins. We demonstrate here that the ERF-associated amphiphilic repression (EAR) motif repression domain (SRDX) can convert a transcriptional complex into a repressor via transrepression that is mediated by protein-protein interactions and show that transrepressive activity of SRDX can be used to detect such protein-protein interactions. When we fused a protein that interacts with a transcription factor with SRDX and co-expressed the product with the transcription factor in plant cells, the expression of genes that are targets of the transcription factor was suppressed by transrepression. We demonstrated the transrepressive activity of SRDX using FOS and JUN as a model system and used two MADS box plant proteins, PISTILLATA and APETALA3, which are known to form heterodimers. Furthermore, the transgenic plants that expressed TTG1, which is a WD40 protein and interacts with bHLH transcription factors, fused to SRDX exhibited a phenotype similar to ttg1 mutants by transrepression and the regions of TTG1 required for interaction to the bHLH protein were detected using our system. We also used this system to analyse a protein factor that might be incorporated into a transcriptional complex and identified an Arabidopsis WD40 protein PWP2 (AtPWP2) interacting with AtTBP1 through comparison of phenotypes induced by 35S:AtPWP2-SRDX with that induced by the chimeric repressor. Our results indicate that the transrepression mediated by SRDX can be used to detect and confirm protein-protein interactions in plants and should be useful in identifying factors that form transcriptional protein complexes.

Isolation and characterization of a novel EAR-motif-containing gene GmERF4 from soybean (Glycine max L.)

Transcriptional repressors are emerging as central regulators of development and stress responses in different organisms. The ERF-associated amphiphilic repression (EAR) motif was identified as essential for transcriptional repression. To gain a better understanding of this type of protein, we reported here a novel GmERF4 protein from soybean. Sequence alignment showed that GmERF4 contains one AP2/ERF domain, two putative nuclear localization signal regions and one EAR motif. The GmERF4 protein was preferentially localized to the nucleus of onion epidermis cells and bound specifically to the GCC box and DRE/CRT element in vitro. Furthermore, the expression of GmERF4 was induced by ethylene, JA, SA, cold, salt, drought, and soybean mosaic virus, and repressed by ABA. Constitutive expression of GmERF4 in transgenic tobacco plants increased tolerance to salt and drought stresses compared with wild-type plants, but did not exhibit detectable resistance against bacterial infection.

A Novel Group of Transcriptional Repressors in Arabidopsis

We showed previously that the ERF-associated amphiphilic repression (EAR) motif is a plant-specific repression domain that contains the conserved amino acid sequence LXLXL. In this report, we describe the identification of a novel repression domain, L/VR/KLFGVXM/V/L, which is different from known EAR motifs, in B3 DNA-binding domain transcription factors in Arabidopsis. Database analysis revealed that 29 Arabidopsis transcription factors, which included members of the RAV, ARF, Hsf and MYB families, contain the R/KLFGV conserved motif found in the novel repression domain. We demonstrated that factors that contain the R/KLFGV motif, namely, RAV1, RAV2, HsfB1 and HsfB2b, exhibited the repressive activity.

AtMYBL2, a protein with a single MYB domain, acts as a negative regulator of anthocyanin biosynthesis in Arabidopsis

In Arabidopsis, MYB transcription factors regulate flavonoid biosynthesis via the formation of protein complexes with a basic helix-loop-helix (bHLH) transcription factor and a WD40 repeat protein. Several R3-type single-MYB proteins (R3-MYB), such as CPC and TRY, act as negative regulators of the development of epidermal cells. However, such regulators of flavonoid biosynthesis have not yet been reported, to our knowledge. We show here that an R3-MYB protein, AtMYBL2, acts as a transcriptional repressor and negatively regulates the biosynthesis of anthocyanin in Arabidopsis. In an AtMYBL2 knockout line (mybl2), the expression of the DFR and TT8 genes was enhanced and resulted in the ectopic accumulation of anthocyanin, while ectopic expression of AtMYBL2 or of a chimeric repressor that is a dominant negative form of AtMYBL2 suppressed the expression of DFR and TT8, and the biosynthesis of anthocyanin. The expression of AtMYBL2 was detected in various tissues but not in those in which anthocyanin accumulated or TT8 was expressed. The minimal repression domain of AtMYBL2 was found to be the six amino acids (TLLLFR) at the carboxyl terminus, and TLLLFR appears to be a novel repression motif that is different from the ERF-associated amphiphilic repression (EAR) motif. The defective phenotype of mybl2 mutants was complemented by 35S:AtMYBL2 but enhanced by a truncated form of AtMYBL2 from which the repression domain had been deleted. AtMYBL2 bound directly to TT8 protein and this complex suppressed the expression of DFR and TT8. The repression activity of AtMYBL2 appears to play a critical role in the regulation of anthocyanin biosynthesis.

Molecular cloning, expression profile and promoter analysis of a novel ethylene responsive transcription factor gene GhERF4 from cotton ( Gossypium hirstum)

Ethylene-responsive element binding factors (ERFs) are plant-specific transcription factors, many of which have been linked to stress responses. A novel ERF gene, designated GhERF4, was isolated by RACE-PCR from Gossypium hirstum. The GhERF4 cDNA has a total length of 1061 bp with an open reading frame of 669 bp, encoding a protein of 222 amino acids with a molecular weight of 23.5 kDa and a calculated p I of 9.03. Sequence alignment shows that GhERF4 contains a 58 amino acid long AP2/ERF domain and a RKRP nuclear localization signal, and belongs to a group II protein in the ERF subfamily as typified by the C-terminal ERF-associated Amphiphilic Repression (EAR) motif. Southern blot analysis indicates that GhERF4 is a single copy gene in cotton genome. Using green fluorescent protein fusion, we demonstrate that GhERF4 accumulates specifically in the nucleus of onion epidermis cells. Semi-quantitative RT-PCR reveals that GhERF4 is constitutively expressed in true leaves, roots, seeds and stems. The transcripts of GhERF4 accumulate highly and rapidly when plants are treated with exogenous ethylene, salt, cold, drought stresses and exogenous abscisic acid (ABA) treatment, suggesting that GhERF4 is regulated by certain components of the stress signaling pathway. Promoter analysis indicates that the 5′ upstream region of GhERF4 possesses some elements induced by physiological and environmental factors. These results indicate that GhERF4 may play an important role in response to ethylene, ABA and environmental stresses.