Ethylene Response Factor Proteins Research Articles

Induction of multiple defense responses in wheat plants against stripe rust using mycorrhizal fungi and Streptomyces viridosporus HH1

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating diseases of wheat worldwide. This study dealt with investigating the biocontrol activity of mycorrhizal colonization and/or application of Streptomyces viridosporus HH1 against stripe rust of wheat. In vitro, data showed that S. viridosporus HH1 had a strong anti-spore germination effect on uredospores of P. striiformis (90% reduction). A 84.2% reduction in the disease severity was achieved in the infected wheat plants, which were colonized with mycorrhizal fungi and were sprayed with S. viridosporus HH1. Molecular investigation revealed that mycorrhizal colonization and application of S. viridosporus HH1 upregulated the defensive genes ethylene response factor protein 3 (JERF3) by 20.7-fold, chitinase II (13.6-fold), and β-1,3-glucanase (9.8-fold). Furthermore, mycorrhizal colonization and application of S. viridosporus HH1 led to a significant increase in the activity of the antioxidant enzymes peroxidase (52.3%), polyphenol oxidase (106.9%), and catalase (87.9%). The phenolic content also increased due to mycorrhizal colonization and application of S. viridosporus HH1, while the lipid peroxidation decreased in the infected wheat leaves. A mitigation in the adverse effects of infection on the photosynthetic pigments in wheat leaves was noticed. Observations from transmission electron microscopy showed that different hypersensitivity reactions were induced on the ultrastructural level in the infected wheat cells due to this treatment. In addition, a significant growth-promoting effect was also reported. It can be concluded that mycorrhizal colonization and spraying with S. viridosporus HH1 have a promising biocontrol activity against stripe rust in wheat. Field evaluation is necessary in the future studies before the use recommendation.

TERF1 Regulates Nuclear Gene Expression Through Chloroplast Retrograde Signals

Ethylene is an important phytohormone that regulates many important biological processes in plant. ERF (ethylene response factor) proteins are key transcription factors that activate the ethylene signaling pathway. However, our knowledge about the mechanism of the ERF transcription factors in regulating nuclear genes expression is limited. Retrograde signaling pathway in chloroplast is a novel kind of mechanism that regulates nuclear gene expression by different signals in plastid. Based on our former research we analyzed the components related with retrograde signaling from plastid in the transgenic tobacco overexpressing TERF1, a member of ERF family transcription factors, to elucidate the interaction between ethylene signaling pathway and different retrograde signaling pathway in plastid under normal growth condition. Results show that TERF1 regulates different retrograde signals in plastid and thus regulates different nuclear genes expression under normal growth condition. We propose a new mechanism that links ethylene signaling pathway and retrograde signaling pathway as well as new potential of TERF1 in regulating nuclear genes expression at post-transcriptional level.

Genome-wide identification and expression analysis reveal the potential function of ethylene responsive factor gene family in response to Botrytis cinerea infection and ovule development in grapes (Vitis vinifera L.).

The prevention of Botrytis cinerea infection and the study of grape seedlessness are very important for grape industries. Finding correlated regulatory genes is an important approach towards understanding their molecular mechanisms. Ethylene responsive factor (ERF) gene family play critical roles in defence networks and the growth of plants. To date, no large-scale study of the ERF proteins associated with pathogen defence and ovule development has been performed in grape (Vitis vinifera L.). In the present study, we identified 113 ERF genes (VvERF) and named them based on their chromosome locations. The ERF genes could be divided into 11 groups based on a multiple sequence alignment and a phylogenetic comparison with homologues from Arabidopsis thaliana. Synteny analysis and Ka/Ks ratio calculation suggested that segmental and tandem duplications contributed to the expansion of the ERF gene family. The evolutionary relationships between the VvERF genes were investigated by exon-intron structure characterisation, and an analysis of the cis-acting regulatory elements in their promoters suggested potential regulation after stress or hormone treatments. Expression profiling after infection with the fungus, B. cinerea, indicated that ERF genes function in responses to pathogen attack. In addition, the expression levels of most ERF genes were much higher during ovule development in seedless grapes, suggesting a role in ovule abortion related to seedlessness. Taken together, these results indicate that VvERF proteins are involved in responses to Botrytis cinerea infection and in grape ovule development. This information may help guide strategies to improve grape production.

Cloning and molecular characterization of TaERF6, a gene encoding a bread wheat ethylene response factor.

Ethylene response factor proteins are important for regulating gene expression under different stresses. Different isoforms for ERF have previously isolated from bread wheat (Triticum aestivum L.) and related genera and called from TaERF1 to TaERF5. We isolated, cloned and molecular characterized a novel one based on TdERF1, an isoform in durum wheat (Triticum turgidum L.) and called TaERF6. Its cDNA was synthesized, sequenced and compared with genomic sequence to figure out intron and exon regions and determine coding sequence region. The length of TdERF1 gene was 1939 bp and cDNA was 1065 bp including two exons, the first one 259 bp and the second one 806 bp separated by a 874 bp intron with a 111 bp 5'-UTR (untranslated region) and 401 bp 3'-UTR. TaERF6 encodes a 353 amino acids protein with nearly 99% identity to TdERF1. Hydrophobic cluster analysis revealed an N-terminal hydrophobic domain contains a highly conserved motif with the consensus sequence of M [C/L/Y] [G/R] [G/R/P] [A/G/V/L/R] [I/L/R/S/P/Q] [L/I/R/H] and hydrophobic clusters in AP2/ERF domain of which tends to form -sheet. Three monopartite nuclear localization signals also identified in TaERF6 that play important role in getting back into the nucleus. The results showed several putative phosphorylation sites in TaERF6 that a motif from residues 246 to 266, the CMVII-4 motif, was predicted to phosphorylate by different kinase proteins and play important roles in TaERF6 function. Phylogenetic analysis showed 7 clusters (I to VII) and 10 subclusters according to their relatedness in Poaceae family.

Gene expression analysis reveals function of TERF1 in plastid-nucleus retrograde signaling under drought stress conditions

Ethylene response factor (ERF) is a key transcription factor of plant ethylene signaling pathway, which plays an important role in plant response to abiotic and biotic stresses by regulating the expression of downstream genes. However, little is known about the mechanisms of the regulation of gene expression by ERF proteins. Chloroplast is an essential organelle that is important for photosynthesis and biosynthesis of many essential metabolites. There exists an interaction between chloroplasts and the nucleus. Chloroplasts can send multiple kinds of signals to regulate the nuclear gene expression known as retrograde signaling. In our study, we have analyzed the expression of the components related to plastid retrograde signaling pathway to elucidate the mechanism of tomato ethylene responsive factor 1 (TERF1) in response to drought stress. Our results showed that TERF1 can regulate different biogenic and operational retrograde signals to regulate nuclear genes expression, which can improve plant tolerance to drought stress. We also propose a new potential of TERF1 in regulating nuclear gene expression, including regulation of different phytohormone signaling pathways and gene posttranscriptional modification triggered by different retrograde signals. Our results have enriched our knowledge about the function of ERF proteins and ethylene signaling pathway.

Ethylene response factor BnERF2-like (ERF2.4) from Brassica napus L. enhances submergence tolerance and alleviates oxidative damage caused by submergence in Arabidopsis thaliana

Ethylene response factor proteins play an important role in regulating a variety of stress responses in plants, but their exact functions in submergence stress are not well understood. In this study, we isolated BnERF2.4 from Brassica napus L. to study its function in submergence tolerance. The expression of the BnERF2.4 gene in B. napus and the expression of antioxidant enzyme genes in transgenic Arabidopsis were analyzed by quantitative RT-PCR. The expression of BnERF2.4 was induced by submergence in B. napus and the overexpression of BnERF2.4 in Arabidopsis increased the level of tolerance to submergence and oxidative stress. A histochemical method detected lower levels of H2O2, O2•− and malondialdehyde (MDA) in transgenic Arabidopsis. Compared to the wild type, transgenic lines also had higher soluble sugar content and higher activity of antioxidant enzymes, which helped to protect plants against the oxidative damage caused by submergence. It was concluded that BnERF2.4 increased the tolerance of plants to submergence stress and may be involved in regulating soluble sugar content and the antioxidant system in defense against submergence stress.

OsERF2 controls rice root growth and hormone responses through tuning expression of key genes involved in hormone signaling and sucrose metabolism.

Root determines plant distribution, development progresses, stress response, as well as crop qualities and yields, which is under the tight control of genetic programs and environmental stimuli. Ethylene responsive factor proteins (ERFs) play important roles in plant growth and development. Here, the regulatory function of OsERF2 involved in root growth was investigated using the gain-function mutant of OsERF2 (nsf2857) and the artificial microRNA-mediated silenced lines of OsERF2 (Ami-OsERF2). nsf2857 showed short primary roots compared with the wild type (WT), while the primary roots of Ami-OsERF2 lines were longer than those of WT. Consistent with this phenotype, several auxin/cytokinin responsive genes involved in root growth were downregulated in nsf2857, but upregulated in Ami-OsERF2. Then, we found that nsf2857 seedlings exhibited decreased ABA accumulation and sensitivity to ABA and reduced ethylene-mediated root inhibition, while those were the opposite in Ami-ERF2 plants. Moreover, several key genes involved in ABA synthesis were downregulated in nsf2857, but unregulated in Ami-ERF2 lines. In addition, OsERF2 affected the accumulation of sucrose and UDPG by mediating expression of key genes involved in sucrose metabolism. These results indicate that OsERF2 is required for the control of root architecture and ABA- and ethylene-response by tuning expression of series genes involved in sugar metabolism and hormone signaling pathways.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-015-0416-9) contains supplementary material, which is available to authorized users.

Isolation and characterization of StERF transcription factor genes from potato (Solanum tuberosum L.)

Ethylene response factor (ERF) is a major subfamily of the AP2/ERF family and plays significant roles in the regulation of abiotic- and biotic-stress responses. ERF proteins can interact with the GCC-box cis-element and then initiate a transcriptional cascade activating downstream ethylene response and enhancing plant stress tolerance. In this research, we cloned five StERF genes from potato (Solanum tuberosum L.). The expressional analysis of StERF genes revealed that they showed tissue- or organ-specific expression patterns and the expression levels in leaf, stem, root, flower, and tuber were different. The assays of quantitative real-time polymerase chain reaction (qRT-PCR) and the reverse transcription-PCR (RT-PCR) showed that the expression of five StERF genes was regulated by ethephon, methyl jasmonate (MeJA), salt and drought stress. The result from the yeast one-hybrid experiment showed that five StERFs had trans-activation activity and could specifically bind to the GCC-box cis-elements. The StERFs responded to abiotic factors and hormones suggested that they possibly had diverse roles in stress and hormone regulation of potato.

Identification of ERF genes in peanuts and functional analysis of AhERF008 and AhERF019 in abiotic stress response.

Ethylene-responsive factor (ERF) play an important role in regulating gene expression in plant development and response to stresses. In peanuts (Arachis hypogaea L.), which produce flowers aerially and pods underground, only a few ERF genes have been identified so far. This study identifies 63 ERF unigenes from 247,313 peanut EST sequences available in the NCBI database. The phylogeny, gene structures, and putative conserved motifs in the peanut ERF proteins were analysed. Comparative analysis revealed the absence of two subgroups (A1 and A3) of the ERF family in peanuts; only 10 subgroups were identified in peanuts compared to 12 subgroups in Arabidopsis and soybeans. AP2/ERF domains were found to be conserved among peanuts, Arabidopsis, and soybeans. Outside the AP2/ERF domain, many soybean-specific conserved motifs were also detected in peanuts. The expression analysis of ERF family genes representing each clade revealed differential expression patterns in response to biotic and abiotic stresses. Overexpression of AhERF008 influenced the root gravity of Arabidopsis, whereas overexpression of AhERF019 enhanced tolerance to drought, heat, and salt stresses in Arabidopsis. The information generated in this study will be helpful to further investigate the function of ERFs in plant development and stress response.

New Role for an Old Rule: N‐end Rule‐Mediated Degradation of Ethylene Responsive Factor Proteins Governs Low Oxygen Response in PlantsF

The N-end rule pathway regulates protein degradation, which depends on exposed N-terminal sequences in prokaryotes and eukaryotes. In plants, conserved and specific enzymes stimulate selective proteolysis. Although a number of developmental and growth phenotypes have been reported for mutants in the N-end rule, its function has remained unrelated to specific physiological pathways. The first report of the direct involvement of the N-end rule in stress responses focused on hypoxic signaling and how the oxygen-dependent oxidation of cystein promotes the N-end rule-mediated degradation of ethylene responsive factor (ERF)-VII proteins, the master regulators of anaerobic responses. It has been suggested that plants have evolved specific mechanisms to tune ERF-VII availability in the nucleus. In this review, we speculate that ERF-VII proteins are reversibly protected from degradation via membrane sequestration. The oxidative response in plants subjected to anoxic conditions suggests that reactive oxygen and nitrogen species (reactive oxygen species and reactive nitrogen species) may interact or interfere with the N-end rule pathway-mediated response to hypoxia.

An alfalfa (Medicago sativa L.) ethylene response factor gene, MsERF11, enhances salt tolerance in transgenic Arabidopsis

A novel orthologue of ethylene response factor gene, MsERF11, was isolated from alfalfa in this study. It has an open reading frame of 807 bp, encoding a predicted polypeptide of 268 amino acids. Sequence similarity analysis clearly suggested that MsERF11 encoded an ethylene response factor protein. The results of transient expression of MsERF11 in onion epidermal cells indicated that MsERF11 is a nuclear protein. The expression pattern of MsERF11 gene was analyzed by real-time quantitative PCR and a higher level of expression was observed in leaves than was observed in roots, stems, flower buds and flowers. Furthermore, the expression was induced by PEG6000, NaCl, Al2(SO4)3 and six different hormones. Over-expressing MsERF11 resulted in enhanced tolerances to salt stress in transgenic Arabidopsis plants. This research indicates that MsERF11 has the potential to be used for improving crop's salt tolerance in areas where salinity is a limiting factor for agricultural productivity. MsERF11 was isolated from alfalfa. Its expression was induced by different abiotic stresses and hormones. Over-expressing MsERF11 resulted in enhanced salt tolerance in transgenic Arabidopsis plants.

An ethylene response factor (ERF5) promoting adaptation to drought and salt tolerance in tomato

A novel member of the AP2/ERF transcription factor family, SlERF5, was identified from a tomato mature leaf cDNA library screen. The complete DNA sequence of SlERF5 encodes a putative 244-amino acid DNA-binding protein which most likely acts as a transcriptional regulator and is a member of the ethylene responsive factor (ERF) superfamily. Analysis of the deduced SlERF5 protein sequence showed that it contained an ERF domain and belonged to the class III group of ERFs proteins. Expression of SlERF5 was induced by abiotic stress, such as high salinity, drought, flooding, wounding and cold temperatures. Over-expression of SlERF5 in transgenic tomato plants resulted in high tolerance to drought and salt stress and increased levels of relative water content compared with wild-type plants. This study indicates that SlERF5 is mainly involved in the responses to abiotic stress in tomato.

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.

An AP2 Domain-Containing Gene, ESE1, Targeted by the Ethylene Signaling Component EIN3 Is Important for the Salt Response in Arabidopsis

Accumulating investigations reveal that ethylene signaling is involved in the salt response in Arabidopsis (Arabidopsis thaliana), and it has been reported that overexpression of a number of ethylene response factor (ERF) genes enhances salt tolerance; however, transcriptional regulation of the ethylene signal component ETHYLENE INSENSITIVE3 (EIN3) in the salt response has not been clearly defined. Consulting microarray data and transcriptional confirmations showed that three of the ERF genes were ethylene and salt inducible, named ESE1 to ESE3. Additionally, the expression of one of the ESE genes (ESE1) was suppressed in ein2, ein3-1, eil1-3, and ein3 eil1 but enhanced in EIN3-overexpressing (EIN3ox) lines. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine, and ethylene action, AgNO₃, reduced the expression of ESE1, while ethylene overproduction eto mutants enhanced the expression of ESE1, indicating that ESE1 is an ethylene-modulated gene downstream of EIN3/EIL1. Further analyses with biochemical and molecular approaches revealed that EIN3 physically binds to the ESE1 promoter, demonstrating that ESE1 was one target of EIN3. ESE1 in turn binds to promoters of salt-related genes, such as RD29A and COR15A. Moreover, either EIN3ox or ESE1ox was sufficient to enhance transcript levels of salt-related genes and salt tolerance. In addition, ESE1ox in ein3 enhanced the salt response during seed germination and seedling development, demonstrating that ESE1 is genetically downstream of EIN3. Thus, the evidence in this report reveals that the transcriptional complex of EIN3-ESE1 is a crucial event in the salt response, thereby connecting the transcriptional regulation of EIN3 and the downstream ERF protein ESE1 in the salt response.

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.

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.

Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis

Increasing numbers of investigations indicate that ethylene response factor (ERF) proteins play important roles in plant stress responses via interacting with GCC box and/dehydration-responsive element/C-repeat to modulate expression of downstream genes, but the detailed regulatory mechanism is not well elucidated. Revealing the modulation pathway of ERF proteins in response to stresses is vital. Previously, we showed that tomato ERF protein TERF2/LeERF2 is ethylene inducible, and ethylene production is suppressed in antisense TERF2/LeERF2 tomatoes, suggesting that TERF2/LeERF2 functions as a positive regulator in ethylene biosynthesis. In this paper, we report that regulation of TERF2/LeERF2 in ethylene biosynthesis is associated with enhanced freezing tolerance of tobacco and tomato. Analysis of gene expression showed that cold slowly induces expression of TERF2/LeERF2 in tomato, implying that TERF2/LeERF2 may be involved in cold response through ethylene modulation. To test the hypothesis, we first observed that overexpressing TERF2/LeERF2 tobaccos not only enhances freezing tolerance via activating expression of cold-related genes, but also significantly reduces electrolyte leakage. In addition, with treatment of ethylene biosynthesis inhibitor or ethylene receptor antagonist, we then showed that blockage of ethylene biosynthesis or the ethylene signaling pathway decreases freezing tolerance of overexpressing TERF2/LeERF2 tobaccos. Moreover, the results from tomatoes showed that overexpressing TERF2/LeERF2 tomatoes enhances while antisense TERF2/LeERF2 transgenic lines decreases freezing tolerance, and application of ethylene precursor 1-aminocyclopropane-1-carboxylic acid restored freezing tolerance of antisense lines. Therefore our results establish that TERF2/LeERF2 enhances freezing tolerance of plants through ethylene biosynthesis and the ethylene signaling pathway.

Molecular diversity and gene expression of cotton ERF transcription factors reveal that group IXa members are responsive to jasmonate, ethylene and Xanthomonas.

Several ethylene-response factor (ERF) transcription factors are believed to play a crucial role in the activation of plant defence responses, but little is known about the relationships between the diversity of this family and the functions of groups or individual ERFs in this process. In this study, 200 ERF genes from the unigene cotton database were identified. Conserved amino acid residues and phylogeny reconstruction using the AP2 conserved domain suggest that the classification into 10 major groups used for Arabidopsis and rice is applicable to the cotton ERF family. Based on in silico studies, we predict that group IX ERF genes in cotton are involved in jasmonate (JA), ethylene (ET) and pathogen responses. To test this hypothesis, we analysed the transcript profiles of the group IXa subfamily in the regulation of specific resistance to Xanthomonas campestris pathovar malvacearum. The expression of four members of group IXa was induced on challenge with X. campestris pv. malvacearum. Furthermore, the expression of several ERF genes of group IXa was induced synergistically by JA in combination with ET, suggesting that the encoded ERF proteins may play key roles in the integration of both signals to activate JA- and ET-dependent responses.

Transcriptional Regulation of the Ethylene Response Factor LeERF2 in the Expression of Ethylene Biosynthesis Genes Controls Ethylene Production in Tomato and Tobacco

Fine-tuning of ethylene production plays an important role in developmental processes and in plant responses to stress, but very little is known about the regulation of ethylene response factor (ERF) proteins in ethylene biosynthesis genes and ethylene production. Identifying cis-acting elements and transcription factors that play a role in this process, therefore, is important. Previously, a tomato (Solanum lycopersicum [f. sp. Lycopersicon esculentum]) ERF protein, LeERF2, an allele of TERF2, was reported to confer ethylene triple response on plants. This paper reports the transcriptional modulation of LeERF2/TERF2 in ethylene biosynthesis in tomato and tobacco (Nicotiana tabacum). Using overexpressing and antisense LeERF2/TERF2 transgenic tomato, we found that LeERF2/TERF2 is an important regulator in the expression of ethylene biosynthesis genes and the production of ethylene. Expression analysis revealed that LeERF2/TERF2 is ethylene inducible, and ethylene production stimulated by ethylene was suppressed in antisense LeERF2/TERF2 transgenic tomato, indicating LeERF2/TERF2 to be a positive regulator in the feedback loop of ethylene induction. Further research showed that LeERF2/TERF2 conservatively modulates ethylene biosynthesis in tobacco and that such regulation in tobacco is associated with the elongation of the hypocotyl and insensitivity to abscisic acid and glucose during germination and seedling development. The effects on ethylene synthesis were similar to those of another ERF protein, TERF1, because TERF1 and LeERF2/TERF2 have overlapping roles in the transcriptional regulation of ethylene biosynthesis in tobacco. Biochemical analysis showed that LeERF2/TERF2 interacted with GCC box in the promoter of NtACS3 and with dehydration-responsive element in the promoter of LeACO3, resulting in transcriptional activation of the genes for ethylene biosynthesis in tomato and tobacco, which is a novel regulatory function of ERF proteins in plant ethylene biosynthesis.

Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar‐related Genes

Ethylene response factor (ERF) proteins are important plant-specific transcription factors. Increasing evidence shows that ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interaction with different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activates a cluster gene expression through interacting with GCC box and dehydration responsive element (DRE), resulting in enhanced sensitivity to abscisic acid (ABA) and tolerance to drought, and dark green leaves of mature plants, indicating that TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpression of TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides information indicating that there are many cis-acting elements, including sugar responsive elements (SURE) and W box, suggesting that TERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reaction amplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Further investigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination, root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment. Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE and activates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response. Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.