Overexpression of the Watermelon Ethylene Response Factor ClERF069 in Transgenic Tomato Resulted in Delayed Fruit Ripening

Watermelon fruit undergoes distinct development stages with dramatic changes during fruit ripening.To date,the molecular mechanics of watermelon ripening remain unclear.Genetic and transcriptome evidences suggested that the ethylene response factor(ERF)gene ClERF069 may be an important candidate factor affecting watermelon fruit ripening.To dissect the roles of ClERF069 in fruit ripening,structure and phylogenetic analysis were performed using the amplified full-length sequence.Normal-ripening watermelon 97103,non-ripening watermelon PI296341-FR and the RIL population were used to analyze ClERF069 expression dynamics and the correlation with fruit ripening indexs.The results indicated that ClERF069 belongs to ERF family group VI and show high homology(83%identity)to melon ERF069-like protein.ClERF069 expression in watermelon flesh was negatively correlated with fruit lycopene content and sugar content during fruit ripening progress.Further transgenic evidences indicated that overexpression of 35S:ClERF069 in tomato noticeably delayed the ripening process up to 5.2 days.Lycopene,β-carotenoid accumulation patterns were altered and ethylene production patterns in transgenic fruits was significantly delayed during fruit ripening.Taken together,watermelon ethylene response factor ClERF069 was concluded to be a negative regulator of fruit ripening.

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.

Phosphorylation of an ethylene response factor by MPK3/MPK6 mediates negative feedback regulation of pathogen-induced ethylene biosynthesis in Arabidopsis

Plants under pathogen attack produce high levels of the gaseous phytohormone ethylene to induce plant defense responses via the ethylene signaling pathway.The 1-aminocyclopropane-1-carboxylate synthase(ACS)is a critical rate-limiting enzyme of ethylene biosynthesis.Transcriptional and post-translational upregulation of ACS2 and ACS6 by the mitogen-activated protein kinases MPK3 and MPK6 are previously shown to be crucial for pathogen-induced ethylene biosynthesis in Arabidopsis.Here,we report that the fungal pathogen Botrytis cinerea-induced ethylene biosynthesis in Arabidopsis is under the negative feedback regulation by ethylene signaling pathway.The ethylene response factor ERF1 A is further found to act downstream of ethylene signaling to negatively regulate the B.cinerea-induced ethylene biosynthesis via indirectly suppressing the expression of ACS2 and ACS6.Interestingly,ERF1 A is shown to also upregulate defensin genes directly and therefore promote Arabidopsis resistance to B.cinerea.Furthermore,ERF1 A is identified to be a substrate of MPK3 and MPK6,which phosphoactivate ERF1 A to enhance its functions in suppressing ethylene biosynthesis and inducing defensin gene expression.Taken together,our data reveal that ERF1 A and its phosphorylation by MPK3/MPK6 not only mediate the negativefeedback regulation of the B.cinerea-induced ethylene biosynthesis,but also upregulate defensin gene expression to increase Arabidopsis resistance to B.cinerea.

Identification and characterization of MeERF genes and their targets in pathogen response by cassava(Manihot esculenta)

Cassava,Manihot esculenta Crantz (Me),is a major dietary source of calories for over 700 million people in tropical regions.The production of cassava is constantly threatened by cassava bacterial blight (CBB),caused by Xanthomonas axonopodis pv.manihotis (Xam).The gene resources for CBB-resistant breeding of cassava are limited.In model plant species,ethylene response factors play important roles in response to pathogen infection.In this study,cassava ethylene response factors (MeERFs) were identified and characterized as the first step in studying their potential for CBB-resistant breeding of cassava.In the cassava genome 155 MeERFs were identified,of which 23 were induced by Xam infection.The promoter regions of204 genes harbored GCC-box that had the potential to interact with MeERFs.Using 37 transcriptomes derived from Xam infection treatment,four gene co-expression modules for the MeERFs and GCC-box containing genes were constructed.Six MeERFs were associated with two GCC-box containing genes:transcription initiation factor TFIIE subunit beta (MeTFIIE),and histone-lysine N-methyltransferase ASHR1 (MeASHR1).Dual-luciferase reporter assays showed that MeERF10 and MeERF58 positively regulated Me TFIIE;MeERF137 negatively regulated Me TFIIE;MeERF10 and MeERF137 positively regulated Me ASHR1;and MeERF35 negatively regulated Me ASHR1.The four MeERFs may mediate pathogen response by regulating the expression of the two GCC-box containing genes.

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

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.

Cotton Major Latex Protein 28 Functions as a Positive Regulator of the Ethylene Responsive Factor 6 in Defense against Verticillium dahliae

In this study, we identified a defense-related major latex protein （MLP） from upland cotton （designated GhMLP28） and investigated its functional mechanism. GhMLP28 transcripts were ubiquitously present in cotton plants, with higher accumulation in the root. Expression of the GhMLP28 gene was induced by Verticillium dahliae inoculation and was responsive to defense signaling molecules, including ethylene, jas- monic acid, and salicylic acid. Knockdown of GhMLP28 expression by virus-induced gene silencing re- sulted in increased susceptibility of cotton plants to V. dahliae infection, while ectopic overexpression of GhMLP28 in tobacco improved the disease tolerance of the transgenic plants. Further analysis revealed that GhMLP28 interacted with cotton ethylene response factor 6 （GhERF6） and facilitated the binding of GhERF6 to GCC-box element. Transient expression assay demonstrated that GhMLP28 enhanced the tran- scription factor activity of GhERF6, which led to the augmented expression of some GCC-box genes. GhMLP28 proteins were located in both the nucleus and cytoplasm and their nuclear distribution was dependent on the presence of GhERF6. Collectively, these results demonstrate that GhMLP28 acts as a positive regulator of GhERF6, and synergetic actions of the two proteins may contribute substantially to protection against V. dahliae infection in cotton plants.

ThERF1 regulates its target genes via binding to a novel cis-acting element in response to salt stress

Ethylene responsive factors (ERFs) are plant-specific transcription factors that are involved in a variety of biological processes. We previously demonstrated that an ERF gene from Tamarix hispida, ThERF1, encodes a protein binding to GCC-box and DRE motifs and negatively modulates abiotic stress tolerance. In the present study, microarray analysis was performed to study the genes regulated by ThERF1 on a genomic scale. There were 154 and 307 genes (respectively representing 134 and 260 unique genes) significantly up- and downregulated by ThERF1 under salt stress conditions, respectively. A novel motif, named TTG, was identified to be recognized by ThERF1, which commonly presents in the promoters of ThERF1-targeted genes. The TTG motif is also bound by other ERFs of a different subfamily from T. hispida and Arabidopsis, indicating that it is commonly recognized by ERF proteins. The binding affinities of ERFs to the TTG motif are significantly induced by salt stress. The TTG motif is more enriched than the GCC-box and DRE motifs in the promoters of ThERF1-targeted genes. Taken together, these studies suggested that the TTG motif plays an important role in the gene expression regulated by ERFs in response to salt stress.

Role of ethylene and proteolytic N-degron pathway in the regulation of Arabidopsis seed dormancy

Primary dormant seeds of Arabidopsis thaliana did not germinate in darkness at temperature higher than 10–15℃.Ethylene improved the germination of dormant wild-type(Col-0)seeds at 25℃ in darkness but seeds of the mutant affected in the proteolytic N-degronpathway,proteolysis6(prt6),were insensitive to ethylene suggesting that PRT6 was involved in dormancy release by ethylene.The substrates of the N-degron pathway,the Ethylene Response Factors from group VII(HRE1,HRE2,RAP2.2,RAP2.3,and RAP2.12),were identified to be involved in this insensitivity with an increased germination in prt6 rap2.2 rap2.3 rap2.12 rather than in prt6 hre1 hre2,which also indicated that the three RAPs acted downstream of PRT6,while the two HREs acted upstream of PRT6.Ethylene reduced the expression of the three RAPs in Col-0 seeds but they were maintained or induced by ethylene in prt6 seeds.The promoting effect of ethylene was associated with a down-regulation of dormancy-related genes in gibberellins(GAs)and abscisic acid(ABA)signaling,such as RGA,RGL2,and ABI5,and with a strong decrease in ABA/GA4 ratio in the presence of ethylene.In contrast,we show that the insensitivity of prt6 seeds to ethylene was mainly related to GA signaling disturbance.

ERF1 delays flowering through direct inhibition of FLOWERING LOCUS T expression in Arabidopsis

ETHYLENE RESPONSE FACTOR1(ERF1)is a key component in ethylene signaling,playing crucial roles in both biotic and abiotic stress responses.Here,we demonstrate that ERF1 also has an important role during floral initiation in Arabidopsis thaliana.Knockdown or knockout of ERF1 accelerated floral initiation,whereas overexpression of ERF1 dramatically delayed floral transition.These contrasting phenotypes were correlated with opposite transcript levels of FLOWERING LOCUS T(FT).Chromatin immunoprecipitation(ChIP)assays revealed that ERF1 associates with genomic regions of the FT gene to repress its transcription.ft-10/ERF1RNAi plants showed a similar flowering phenotype to the ft-10 mutant,whereas the flowering of FTox/ERF1ox mimicked that of FTox plants,suggesting that ERF1 acts upstream of FT during floral initiation.Similarly,altered floral transition in ethylene-related mutants was also correlated with FT expression.Further analysis suggested that ERF1 also participates in delay in flowering-time control mediated by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid.Thus,ERF1 may act as a negative modulator of flowering-time control by repressing FT transcription in Arabidopsis.