GCC Box Research Articles

In silico targeted genome mining and comparative modelling reveals a putative protein similar to an Arabidopsis drought tolerance DNA binding transcription factor in Chromosome 6 of Sorghum bicolor genome

Arabidopsis Thaliana HARDY (AtHRD) is a gene with an APETELA 2 / Ethylene Responsive Factor (AP2/ERF) domain linked to improved performance under drought in rice. We hypothesized that the sorghum genome could possess a similar gene product and were motivated to conduct a computational genome scale mining for the protein and analyse its structural and functional properties. AtHRD sequence was used as a query to BLAST against the sorghum genome dataset followed by multiple alignment analysis. A homology model of the target was built using a template detected based on the pair-wise comparison of hidden Markov models for alignments. DNA docking with a matrix of homologous interface contacts was done. Functional and structural analysis of the query and target was conducted using various online servers. A High-scoring segment pair from Chromosome 6 of the sorghum genome in the region between 54948120 and 54948668 had 68 amino acid similarities out of the 184 residues and was 1.4% above twilight zone threshold. The homology model showed 86.8% residues in most favoured regions. The target protein which had an AP2/ERF domain when docked with GCC box DNA motif had conserved residues involved in binding; it had a long unstructured region beyond the AP2 domain with several motifs for the recognition of serine/threonine protein kinase group. The protein model showed that it could bind to a GCC box which is present in several drought responsive genes. The presence of possible signalling domains and intrinsic disorder in the target protein suggest that this could play a role in drought tolerance which is an inherent character of sorghum. These results offer a jumpstart for validation experiments which could pave the way for cis/trans genic improvement of a range of crops.

ERF5 and ERF6 Play Redundant Roles as Positive Regulators of JA/Et-Mediated Defense against Botrytis cinerea in Arabidopsis

The ethylene response factor (ERF) family in Arabidopsis thaliana comprises 122 members in 12 groups, yet the biological functions of the majority remain unknown. Of the group IX ERFs, the IXc subgroup has been studied the most, and includes ERF1, ERF14 and ORA59, which play roles in plant innate immunity. Here we investigate the biological functions of two members of the less studied IXb subgroup: ERF5 and ERF6. In order to identify potential targets of these transcription factors, microarray analyses were performed on plants constitutively expressing either ERF5 or ERF6. Expression of defense genes, JA/Et-responsive genes and genes containing the GCC box promoter motif were significantly upregulated in both ERF5 and ERF6 transgenic plants, suggesting that ERF5 and ERF6 may act as positive regulators of JA-mediated defense and potentially overlap in their function. Since defense against necrotrophic pathogens is generally mediated through JA/Et-signalling, resistance against the fungal necrotroph Botrytis cinerea was examined. Constitutive expression of ERF5 or ERF6 resulted in significantly increased resistance. Although no significant difference in susceptibility to B. cinerea was observed in either erf5 or erf6 mutants, the erf5 erf6 double mutant showed a significant increase in susceptibility, which was likely due to compromised JA-mediated gene expression, since JA-induced gene expression was reduced in the double mutant. Taken together these data suggest that ERF5 and ERF6 play positive but redundant roles in defense against B. cinerea. Since mutual antagonism between JA/Et and salicylic acid (SA) signalling is well known, the UV-C inducibility of an SA-inducible gene, PR-1, was examined. Reduced inducibilty in both ERF5 and ERF6 constitutive overexepressors was consistent with suppression of SA-mediated signalling, as was an increased susceptibility to avirulent Pseudomonas syringae. These data suggest that ERF5 and ERF6 may also play a role in the antagonistic crosstalk between the JA/Et and SA signalling pathways.

The rice ERF transcription factor OsERF922 negatively regulates resistance to Magnaporthe oryzae and salt tolerance.

Rice OsERF922, encoding an APETELA2/ethylene response factor (AP2/ERF) type transcription factor, is rapidly and strongly induced by abscisic acid (ABA) and salt treatments, as well as by both virulent and avirulent pathovars of Magnaporthe oryzae, the causal agent of rice blast disease. OsERF922 is localized to the nucleus, binds specifically to the GCC box sequence, and acts as a transcriptional activator in plant cells. Knockdown of OsERF922 by means of RNAi enhanced resistance against M. oryzae. The elevated disease resistance of the RNAi plants was associated with increased expression of PR, PAL, and the other genes encoding phytoalexin biosynthetic enzymes and without M. oryzae infection. In contrast, OsERF922-overexpressing plants showed reduced expression of these defence-related genes and enhanced susceptibility to M. oryzae. In addition, the OsERF922-overexpressing lines exhibited decreased tolerance to salt stress with an increased Na+/K+ ratio in the shoots. The ABA levels were found increased in the overexpressing lines and decreased in the RNAi plants. Expression of the ABA biosynthesis-related genes, 9-cis-epoxycarotenoid dioxygenase (NCED) 3 and 4, was upregulated in the OsERF922-overexpressing plants, and NCED4 was downregulated in the RNAi lines. These results suggest that OsERF922 is integrated into the cross-talk between biotic and abiotic stress-signalling networks perhaps through modulation of the ABA levels.

Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis

One of the most rapid and effective defensive mechanisms plants have for protecting themselves, from a variety of biotic and abiotic stresses, is the regulation of plant signal transcription factors. AP2/ERF factors play an important role in plant development as well as in hormonal regulation and cold response. Directed evolution is a powerful tool to modify proteins, improving their properties, and for studying their structure-function relations. Here, the transgenic Arabidopsis plants over-expressed a mutant gene, BnaERF-B3-hy15-mu3, which encoded for a factor that exhibited more binding activity with the GCC box element than the wild-type gene BnaERF-B3-hy15 encode factor, and exhibited more freezing tolerance than transgenic plants containing the original BnaERF-B3-hy15 gene. Real-time PCR analyses also revealed that the expression levels of several stress-regulated genes were altered in the over-expressed BnaERF-B3-hy15-mu3 transgenic lines. The BnaERF-B3-hy15 responded to exogenous ABA. Using RT-PCR analysis, the expression of BnaERF-B3-hy15 at different stages and stress treatments were also analyzed.

Functional Analysis on GCC Box and JERE Box Mediating Signal Pathway inRice Response to Stress

Plants respond to stresses are mainly regulated by transcriptional modulation.And it is vital that transcription factors recognize and bind to their corresponding cis-elements in the promoters of target genes.In this study,two synthetic promoters were constructed by combining GCC box(ET responsive) or JERE box(JA and elicitor responsive) with the 35S minimal promoter of cauliflower mosaic virus,respectively.The promoters were further cloned into expression vectors to drive β-glucuronidase(GUS) reporter gene and the vectors were transformed into rice via an Agrobacterium-mediated transformation method.The expression of GUS in T1 transgenic lines under different stimuli was analyzed.The results showed that two synthetic promoters had very low background expression.GUS expression could be improved to varying degree by rice blast infection,Cnaphalocrocis medialis biting and mechanical wounding.Moreover,GUS expression could also be improved by ABA,SA and JA.The results suggested a conservation of GCC and JERE mediating defense signaling pathways between dicotyledon and monocotyledon.Fig 5,Ref 15

An ethylene response factor OsWR1 responsive to drought stress transcriptionally activates wax synthesis related genes and increases wax production in rice

Increasing evidence has revealed the major enzymes-involved in Arabidopsis and maize wax/cutin synthesis; however, there is limited information about the genes-associated with wax/cutin synthesis in rice. Here we report the characterization of an ethylene response factor gene in rice. This rice wax synthesis regulatory gene 1 (OsWR1) is a homolog of Arabidopsis wax/cutin synthesis regulatory gene WIN1/SHN1. Transcript analysis showed that OsWR1 is induced by drought, abscisic acid and salt, and is predominantly expressed in leaves. Functional analyses indicated that overexpressing OsWR1 (Ox-WR1) improved while RNA interference OsWR1 rice (RI-WR1) decreased drought tolerance, consistent with water loss and cuticular permeability, suggesting that OsWR1-triggered drought response might be associated with cuticular characteristics. In addition, OsWR1 activated the expression of the genes-related to oxidative stress response and membrane stability. Gas chromatograph-mass spectrometry analysis further showed that OsWR1 modulated the wax synthesis through alteration of long chain fatty acids and alkanes, evidencing the regulation of OsWR1 in wax synthesis. Detection with real-time PCR amplification indicated that Ox-WR1 enhanced while RI-WR1 decreased the expression of wax/cutin synthesis related genes. Furthermore, OsWR1 physically interacted with the DRE and GCC box in the promoters of wax related genes OsLACS2 and OsFAE1'-L, indicating that OsWR1 at least directly modulates the expression of these genes. Thus our results indicate that OsWR1 is a positive regulator of wax synthesis related genes in rice, and this regulation, distinct from its homology regulator of WIN1/SHN1 in cutin synthesis, subsequently contributes to reduced water loss and enhanced drought tolerance.

Arabidopsis RGLG2, Functioning as a RING E3 Ligase, Interacts with AtERF53 and Negatively Regulates the Plant Drought Stress Response

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.

Expression of Arabidopsis SHORT INTERNODES/STYLISH family genes in auxin biosynthesis zones of aerial organs is dependent on a GCC box-like regulatory element.

Auxin/indole-3-acetic acid (IAA) biosynthesis in Arabidopsis (Arabidopsis thaliana) plays a major role in growth responses to developmental and genetic signals as well as to environmental stimuli. Knowledge of its regulation, however, remains rudimentary, and few proteins acting as transcriptional modulators of auxin biosynthesis have been identified. We have previously shown that alteration in the expression level of the SHORT INTERNODES/STYLISH (SHI/STY) family member STY1 affects IAA biosynthesis rates and IAA levels and that STY1 acts as a transcriptional activator of genes encoding auxin biosynthesis enzymes. Here, we have analyzed the upstream regulation of SHI/STY family members to gain further insight into transcriptional regulation of auxin biosynthesis. We attempted to modulate the normal expression pattern of STY1 by mutating a putative regulatory element, a GCC box, located in the proximal promoter region and conserved in most SHI/STY genes in Arabidopsis. Mutations in the GCC box abolish expression in aerial organs of the adult plant. We also show that induction of the transcriptional activator DORNRÖSCHEN-LIKE (DRNL) activates the transcription of STY1 and other SHI/STY family members and that this activation is dependent on a functional GCC box. Additionally, STY1 expression in the strong drnl-2 mutant or the drn drnl-1 puchi-1 triple mutant, carrying knockdown mutations in both DRNL and its close paralogue DRN as well as one of their closest homologs, PUCHI, was significantly reduced, suggesting that DRNL regulates STY1 during normal plant development and that several other genes might have redundant functions.

Function of Gene OsASIE1 in Response to Abiotic Stress in Rice

AP2/EREBP transcription factors play an important role in plant development, hormone response, and responses to biotic and abiotic stress. OsASIE1, a member of the EREBP subfamily of AP2/EREBP transcription factors in rice (Oryza sativa L.), is determined to be involved in abiotic stress response. OsASIE1 expression was induced by drought and salt stresses. In addition, an electrophoresis mobility shift assay (EMSA) revealed that the AP2 domain of the OsASIE1 protein can bind to both DRE (dehydration-responsive element) and GCC box (ethylene response element, ERE) in vitro. All these results indicate that OsASIE1 may participate in abiotic stress response by regulating the expression of downstream genes with DRE and GCC box binding. Overexpression of OsASIE1 in transgenic rice plants might be used to improve tolerance to salt stress.

Transcriptional Activation of OsDERF1 in OsERF3 and OsAP2-39 Negatively Modulates Ethylene Synthesis and Drought Tolerance in Rice

The phytohormone ethylene is a key signaling molecule that regulates a variety of developmental processes and stress responses in plants. Transcriptional modulation is a pivotal process controlling ethylene synthesis, which further triggers the expression of stress-related genes and plant adaptation to stresses; however, it is unclear how this process is transcriptionally modulated in rice. In the present research, we report the transcriptional regulation of a novel rice ethylene response factor (ERF) in ethylene synthesis and drought tolerance. Through analysis of transcriptional data, one of the drought-responsive ERF genes, OsDERF1, was identified for its activation in response to drought, ethylene and abscisic acid. Transgenic plants overexpressing OsDERF1 (OE) led to reduced tolerance to drought stress in rice at seedling stage, while knockdown of OsDERF1 (RI) expression conferred enhanced tolerance at seedling and tillering stages. This regulation was supported by negative modulation in osmotic adjustment response. To elucidate the molecular basis of drought tolerance, we identified the target genes of OsDERF1 using the Affymetrix GeneChip, including the activation of cluster stress-related negative regulators such as ERF repressors. Biochemical and molecular approaches showed that OsDERF1 at least directly interacted with the GCC box in the promoters of ERF repressors OsERF3 and OsAP2-39. Further investigations showed that OE seedlings had reduced expression (while RI lines showed enhanced expression) of ethylene synthesis genes, thereby resulting in changes in ethylene production. Moreover, overexpression of OsERF3/OsAP2-39 suppressed ethylene synthesis. In addition, application of ACC recovered the drought-sensitive phenotype in the lines overexpressing OsERF3, showing that ethylene production contributed to drought response in rice. Thus our data reveal that a novel ERF transcriptional cascade modulates drought response through controlling the ethylene synthesis, deepening our understanding of the regulation of ERF proteins in ethylene related drought response.

High‐throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function

Genomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, molecular tools are required to characterize this ORFeome functionally. A high-throughput microtiter plate-based protoplast transactivation (PTA) system has been established that can be used in a screening approach to define which transcription factor (TF) regulates a given promoter in planta. Using to this procedure, the transactivation properties of 96 TFs can be analyzed rapidly, making use of promoter:Luciferase (LUC)-reporters and luciferase imaging. Applying GATEWAY® technology, we have established a platform to assay more than 700 Arabidopsis TFs. As a proof-of-principle, the ethylene response factor (ERF) family has been studied to evaluate this system. Importantly, distinct subsets of related ERF factors were found to activate specifically the well described target promoters RD29A and PDF1.2 that are under control of DRE or GCC box cis-elements, respectively. Furthermore, several applications of the PTA system have been demonstrated, such as analysis of transcriptional repressors, salt-inducible gene expression or functional interaction of signaling molecules like kinases and TFs. This novel molecular tool will improve functional studies on transcriptional regulation in plants significantly.

Isolation and characterization of a gene encoding an ethylene responsive factor protein from Ceratoides arborescens

Ethylene responsive factor (ERF) proteins play important roles in plant growth and development and regulate biotic and abiotic stress responses. In this study, a full length mRNA encoding a novel ERF-type transcription factor namely Ceratoides arborescens ERF protein (CeERF) was isolated from C. arborescens. The deduced amino acid of CeERF had a conserved APETALA2/ERF (AP2/ERF) domain which specifically binds to cis-acting elements GCC box. Under normal conditions, the expression level of CeERF was highest in leaves and lowest in roots. CeERF expression was induced by 20% PEG in a time-dependent pattern and peaked at 8 h. CeERF also acts in salt- and hormones-induced stresses. Transient expression analysis in onion epidermal cells indicated that CeERF protein localized to nucleus. Overexpression of CeERF in transgenic tobacco plants resulted in higher tolerance to abiotic stresses than in control plants. These results suggested that CeERF might play a role in abiotic stress signal transduction and that overexpression of CeERF might serve as a feasible approach to enhance resistance in forage, even crop.

Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis

Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-010-9728-y) contains supplementary material, which is available to authorized users.

Phytochrome-regulated EBL1 contributes to ACO1 upregulation in rice

The 1-aminocyclopropane-1-carboxylate oxidase gene (ACO1) was upregulated in rice (Oryza sativa L.) phyAphyBphyC mutants lacking any phytochrome and containing the GCC box element, a binding site for rice ethylene-responsive element binding protein 1 (OsEREBP1), in its promoter region. Since the OsEREBP1-like gene EBL1 (OsEREBP1-LIKE 1) was significantly downregulated in phyAphyBphyC mutants, EBL1 was suspected to repress ACO1 expression in wild-type plants. However, ACO1 was downregulated in EBL1 RNA interference plants, and the total length of these plants was slightly shorter than that of wild-type plants. This study shows that EBL1 is positively regulated by phytochrome B and associated with ACO1 upregulation.

Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

In this study, we cloned the ERF-B3 subfamily transcription factor gene BnaERF-B3-hy15 from Brassica napus L. Huyou15. This 600 bp gene encodes a 199 amino acid classic ethylene responsive factor (ERF), which shown no binding or very weak binding GCC box-binding activity by the yeast one-hybrid assay. We used gene shuffling and the yeast one-hybrid system to obtain three mutated sequences that can bind to the GCC box. Sequence analysis indicated that two residues, Gly156 in the AP2 domain and Phe62 at the N-terminal domain were mutated to arginine and serine, respectively. Changes of Gly156 to arginine and Phe62 to serine increased the GCCbinding activity of BnaERF-B3-hy15 and the alter of Gly156 to arginine changed the AP2-domain structure of BnaERF-B3- hy15.

Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution

The B-3 Ethylene Response Factor MtERF1-1 Mediates Resistance to a Subset of Root Pathogens inMedicago truncatulawithout Adversely Affecting Symbiosis with Rhizobia

The fungal necrotrophic pathogen Rhizoctonia solani is a significant constraint to a range of crops as diverse as cereals, canola, and legumes. Despite wide-ranging germplasm screens in many of these crops, no strong genetic resistance has been identified, suggesting that alternative strategies to improve resistance are required. In this study, we characterize moderate resistance to R. solani anastomosis group 8 identified in Medicago truncatula. The activity of the ethylene- and jasmonate-responsive GCC box promoter element was associated with moderate resistance, as was the induction of the B-3 subgroup of ethylene response transcription factors (ERFs). Genes of the B-1 subgroup showed no significant response to R. solani infection. Overexpression of a B-3 ERF, MtERF1-1, in Medicago roots increased resistance to R. solani as well as an oomycete root pathogen, Phytophthora medicaginis, but not root knot nematode. These results indicate that targeting specific regulators of ethylene defense may enhance resistance to an important subset of root pathogens. We also demonstrate that overexpression of MtERF1-1 enhances disease resistance without apparent impact on nodulation in the A17 background, while overexpression in sickle reduced the hypernodulation phenotype. This suggests that under normal regulation of nodulation, enhanced resistance to root diseases can be uncoupled from symbiotic plant-microbe interactions in the same tissue and that ethylene/ERF regulation of nodule number is distinct from the defenses regulated by B-3 ERFs. Furthermore, unlike the stunted phenotype previously described for Arabidopsis (Arabidopsis thaliana) ubiquitously overexpressing B-3 ERFs, overexpression of MtERF1-1 in M. truncatula roots did not show adverse effects on plant development.

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.

Expression of ethylene response factor JERF1 in rice improves tolerance to drought

Ethylene response factor (ERF) proteins regulate a variety of stress responses in plant. JERF1, a tomato ERF protein, can be induced by abscisic acid (ABA). Overexpression of JERF1 enhanced the tolerance of transgenic tobacco to high salt concentration, osmotic stress, and low temperature by regulating the expression of stress-responsive genes by binding to DRE/CRT and GCC-box cis-elements. In this research, we further report that overexpression of JERF1 significantly enhanced drought tolerance of transgenic rice. The overexpression activated the expression of stress-responsive genes and increased the synthesis of the osmolyte proline by regulating the expression of OsP5CS, encoding the proline biosynthesis key enzyme deltal-pyrroline-5-carboxylate synthetase. JERF1 also activated the expression of two ABA biosynthesis key enzyme genes, OsABA2 and Os03g0810800, and increased the synthesis of ABA in rice. Analysis of cis-elements of JERF1-targeted genes pointed to the existence of DRE/CRT and/or GCC box in their promoters, indicating that JERF1 could activate the expression of related genes in rice by binding to these cis-elements. Unlike some other ERF proteins, constructive overexpression of JERF1 did not change the growth and development of transgenic rice, which makes JEFR1 a potentially useful source in breeding for greater tolerance to abiotic stress.

Molecular Characterization of Three Ethylene Responsive Element Binding Factor Genes from Cotton

Ethylene-responsive factors (ERFs) are important regulators of plant gene expression. In this study, three novel ERF genes, GhERF2, GhERF3 and GhERF6, were isolated from cotton (Gossypium hirstum) using rapid amplification of cDNA ends-polymerase chain reaction. Transient expression analysis using GhERF-green fluorescent protein fusions showed that these three proteins were targeted to the nucleus. Fusion proteins consisting of GhERF2, GhERF3 or GhERF6 coupled to the GAL4 DNA binding domain strongly activated transcription in yeast. Furthermore, GhERF6 was shown to be able to bind specifically to GCC boxes using a particle bombardment assay in tobacco cells. Semi-quantitative reverse transcription-polymerase chain reaction revealed that GhERF2 and GhERF3 are constitutively expressed in all organs, while GhERF6 is only constitutively expressed in vegetative organs. When plants were treated with ethylene, abscisic acid, salt, cold and drought, the transcripts of GhERF2, GhERF3 and GhERF6 were rapidly induced to high levels. Promoter analysis also indicated that the 5' upstream regions of the three genes possess elements induced by these physiological and environmental factors. Collectively, our data suggest that GhERF2, GhERF3 and GhERF6 might function as positive trans-acting factors in the plant responses to ethylene, abscisic acid and other stresses and provide useful clues for further research into the mechanism of them in regulating cotton multiple stress responses.