Ethylene Response Factor Transcription Factor Research Articles

ERF transcription factors govern anthocyanin biosynthesis in litchi pericarp by modulating the expression of anthocyanin biosynthesis genes

Anthocyanins are the most prevalent phytochemicals that color the fruits. Ethylene promotes fruit ripening and modulates anthocyanin biosynthesis in plants, however, the underlying molecular mechanism in litchi is unclear. Herein, the slow-reddening cultivar ‘Feizixiao’ (FXZ) and swift-reddening cultivar ‘Guiwei’ (GW) were selected to study the regulation of anthocyanin biosynthesis in litchi. Total anthocyanin contents were found to be higher in ‘GW’ and lower in ‘FZX’. We then comprehensively investigated ethylene response factor (ERF) subfamily genes and their expression patterns in different tissues and fruit developmental stages. Using transcriptome data, eight LcERF genes were identified as putative key genes associated with litchi pericarp coloration and anthocyanin accumulation. To test whether these genes are expressed under ethephon treatment, we applied 100 and 200 µL L−1 ethephon 4 weeks before harvest. Ethephon treatment dramatically increased anthocyanin accumulation as well as the expression of targeted ERF genes. LcERF1/22/25/37 were significantly upregulated in response to ethephon treatment, while LcERF64 was significantly downregulated. Anthocyanin content and expression of anthocyanin-related genes were considerably reduced in LcERF1/22/25/37-silenced fruits, but the opposite trend was observed in LcERF64-silenced fruits. Yeast-one-hybrid and dual luciferase assays revealed that the tested ERFs bind to the promoters of key anthocyanin biosynthesis genes and alter their expression. These findings suggested that ethylene promotes anthocyanin accumulation and consequently fruit coloration by stimulating the positive anthocyanin biosynthesis regulators LcERF1/22/25/37, or by suppressing the negative regulator LcERF64, which regulates the expression LcDFR and LcMYB1 by directly binding to their promoters.

FaERF2 activates two β-1,3-glucanase genes to enhance strawberry resistance to Botrytis cinerea

Ethylene response factor (ERF) is a class of plant-specific transcription factors that play an important role in plant growth, development, and stress response. However, the underlying mechanism of strawberry ERFs in pathogenic responses against Botrytis cinerea (B. cinerea) remains largely unclear. In this study, we isolated FaERF2, a nucleus-localized ERF transcription factor from Fragaria x ananassa. Transiently overexpressing FaERF2 in strawberry fruits significantly enhances their resistant ability to B. cinerea, while silencing FaERF2 in strawberry fruits enhances their susceptibility to B. cinerea. In addition, we found that FaERF2 could directly bind to the cis-acting element GCC box in the promoters of two β-1,3-glucanase genes, FaBG-1 and FaBG-2, and activate their expression. Finally, both strawberry fruits transient expression followed B. cinerea inoculation assays and recombinant protein incubation tests collectively substantiated the inhibitory effect of FaBG-1 and FaBG-2 on B. cinerea mycelium growth. These results revealed the molecular regulation mechanism of FaERF2 in response to B. cinerea and laid foundations for creating disease-resistance strawberry cultivar through genome editing approach.

Involvement of BcERF109 in 1-MCP-delayed yellowing of pak choi (Brassica rapa subsp. Chinensis) via mitigation of major chlorophyll-degrading gene expression during storage

Leaf yellowing is a typical senescence symptom in postharvest pak choi (Brassica rapa subsp. Chinensis). Ethylene response factors (ERFs) are considered important regulators of leaf senescence. This study explored the likely regulatory effects of BcERF on pak choi yellowing via 1-methylcyclopropene (1-MCP) treatments. We identified an ERF transcription factor (TF), BcERF109, which may be involved in ethylene-induced pak choi yellowing. Treatment with 5.0 µL L–1 1-MCP suppressed the transcripts of chlorophyll catabolism genes (BcPPH1, BcRCCR, BcSGR2, and BcNYC1), inhibited decreases in chlorophyll content and chlorophyll fluorescence parameters, delayed chloroplast disintegration, and eventually mitigated yellowing. The treatment also blocked ethylene production and impeded the activities and transcripts of ethylene synthesis enzymes (ACC oxidase [ACO] and 1-aminocyclopropene-1-carboxylate [ACC] synthase [ACS]). In contrast, treatment with 10.0 µL L–1 ethylene accelerated pak choi yellowing, as indicated by greater ethylene production, activities and transcripts of ACO and ACS, and expression of chlorophyll catabolism genes, as well as reductions in chlorophyll content and fluorescence parameters. BcERF109 was identified as a nuclear-localised protein; its transcription levels were downregulated by 1-MCP and upregulated by ethylene. Molecular biology assays revealed that BcERF109 enhanced the transcriptional capacities of BcPPH1, BcRCCR, BcNYC1, and BcSGR2 by binding to their promoters. The transient overexpressing of BcERF109 in tobacco leaves stimulated the expression of endogenous genes (BcPPH1, BcRCCR, BcNYC1, BcSGR2, and BcERF109), leading to accelerated chlorophyll degradation and leaf yellowing. The application of 1-MCP mitigated pak choi yellowing by influencing ethylene synthesis and modulating the regulatory effects of BcERF019 on genes associated with chlorophyll catabolism. These results revealed a new regulatory mechanism involving ERF TFs in 1-MCP-induced delayed yellowing of green vegetables.

Ethylene response factor ERF022 is involved in regulating Arabidopsis root growth.

Ethylene response factors (ERFs) are involved in the regulation of plant development processes and stress responses. In this study, we provide evidence for the role of ERF022, a member of the ERF transcription factor group III, in regulating Arabidopsis root growth. We found that ERF022-loss-of-function mutants exhibited increased primary root length and lateral root numbers, and also morphological growth advantages compared to wild-type. Further studies showed that mutants had enhanced cell size in length in the root elongation zones. These results were accompanied by significant increase in the expression of cell elongation and cell wall expansion related genes SAUR10, GASA14, LRX2, XTH19 in mutants. Moreover, ERF022-mediated root growth was associated with the enhanced endogenous auxin and gibberellins levels. Our results suggest that loss-of-function of ERF022 up-regulated the expression of cell elongation and cell wall related genes through auxin and gibberellins signal in the regulation of root growth. Unexpectedly, ERF022 overexpression lines also showed longer primary roots and more lateral roots compared to wild-type, and had longer root apical meristematic zone with increased cell numbers. Overexpression of ERF022 significantly up-regulated cell proliferation, organ growth and auxin biosynthesis genes EXO, HB2, GALK2, LBD26, YUC5, which contribute to enhanced root growth. Altogether, our results provide genetic evidence that ERF022 plays an important role in regulating root growth in Arabidopsis thaliana.

Citrus ACC synthase CiACS4 regulates plant height by inhibiting gibberellin biosynthesis.

Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4-CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4-CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

Genome-Wide Identification of the ERF Transcription Factor Family for Structure Analysis, Expression Pattern, and Response to Drought Stress in Populus alba × Populus glandulosa.

The Ethylene Responsive Factor (ERF) transcription factor family is important for regulating plant growth and stress responses. Although the expression patterns of ERF family members have been reported in many plant species, their role in Populus alba × Populus glandulosa, an important model plant for forest research, remains unclear. In this study, we identified 209 PagERF transcription factors by analyzing the P. alba × P. glandulosa genome. We analyzed their amino acid sequences, molecular weight, theoretical pI (Isoelectric point), instability index, aliphatic index, grand average of hydropathicity, and subcellular localization. Most PagERFs were predicted to localize in the nucleus, with only a few PagERFs localized in the cytoplasm and nucleus. Phylogenetic analysis divided the PagERF proteins into ten groups, Class I to X, with those belonging to the same group containing similar motifs. Cis-acting elements associated with plant hormones, abiotic stress responses, and MYB binding sites were analyzed in the promoters of PagERF genes. We used transcriptome data to analyze the expression patterns of PagERF genes in different tissues of P. alba × P. glandulosa, including axillary buds, young leaves, functional leaves, cambium, xylem, and roots, and the results indicated that PagERF genes are expressed in all tissues of P. alba × P. glandulosa, especially in roots. Quantitative verification results were consistent with transcriptome data. When P. alba × P. glandulosa seedlings were treated with 6% polyethylene glycol 6000 (PEG6000), the results of RT-qRCR showed that nine PagERF genes responded to drought stress in various tissues. This study provides a new perspective on the roles of PagERF family members in regulating plant growth and development, and responses to stress in P. alba × P. glandulosa. Our study provides a theoretical basis for ERF family research in the future.

The transcription factor HcERF4 confers salt and drought tolerance in kenaf (Hibiscus cannabinus L.)

Ethylene response factors (ERF) are members of the APETALA2/ERF transcription factor family, and they play an important role in plant growth, development, and response to various environmental stresses. In this study, an ERF transcription factor HcERF4 was isolated and characterized from kenaf. The results showed HcERF4 contained an open reading frame of 702 bp, which comprised of single AP2 domain. Subcellular localization analysis revealed that the HcERF4 gene was localized in the nucleus and cytoplasm. Transactivation assays in yeast demonstrated that HcERF4 functions as a transcriptional activator. Its expression profile was investigated by real-time PCR analysis in different tissues and under salt as well as drought stress condition. The result revealed that HcERF4 could be markedly induced by salt and drought in a time and concentration dependent manner. Moreover, silencing of HcERF4 by using the VIGS technique, caused a significant growth inhibition under salt or drought stress condition, with lower plant height, stem diameter, and fresh weight, indicating HcERF4 plays key roles response abiotic stress. In addition, physiological index analyses showed that the contents of MDA, O2−, H2O2, and free proline significantly increased, while the activities of antioxidant enzymes were significantly restrained in HcERF4 silenced plants. Furthermore, the expression level of eight important genes involved in plant growth regulation, including HcERF3, HcNAC29, HcSOD, HcP5CS, HcbZIP43, HcWRKY31, HcMYB44, and HcCBP25 were investigated, and the result showed that all eight genes were highly expressed in the leaves of the wild kenaf, but were significantly downregulated in the leaves of the VIGS kenaf plants under drought and salt stress conditions; Consequently, the present results indicate HcERF4 plays a positive role in regulating salt and drought stress in kenaf.

The wheat SHORT ROOT LENGTH 1 gene TaSRL1 controls root length in an auxin-dependent pathway.

The root is the main organ for water and nutrient uptake and sensing environmental stimuli in the soil. The optimization of root system architecture contributes to stress tolerance and yield improvement. ERF (ETHYLENE RESPONSIVE FACTOR) is one of the plant-specific transcription factor families associated with various developmental processes and stress tolerance. We cloned a novel ERF transcription factor gene TaSRL1 (SHORT ROOT LENGTH 1) from wheat (Triticum aestivum) which is mainly expressed in root. Ectopic expression of TaSRL1 in rice resulted in short root length and plant height. TaSRL1 regulated expression of genes related to auxin synthesis, transport, and signaling. Further studies revealed that TaSRL1 induced expression of the auxin transport gene TaPIN2 by directly binding to its promoter, while the interaction of TaSRL1 and TaTIFY9 repressed TaPIN2 expression. Sequence polymorphisms and association analysis showed that TaSRL1-4A was associated with root depth and angle, plant height, and 1000-grain weight of wheat. The haplotype Hap-4A-2 with shallow roots, short plant height, and high 1000-grain weight has been positively selected in the Chinese wheat breeding process. We demonstrated that TaSRL1 functions as a direct regulator of TaPIN2 in the auxin-dependent pathway, and integrates auxin and jasmonate signaling by interacting with TaTIFY9 to repress root growth. Furthermore, the molecular marker of TaSRL1-4A is valuable for the improvement of the root system, plant architecture, and yield in the wheat breeding process.

Maize Ethylene Response Factor ZmERF061 Is Required for Resistance to Exserohilum turcicum.

Plants have evolved a series of sophisticated defense mechanisms to help them from harm. Ethylene Response Factor (ERF) plays pivotal roles in plant immune reactions, however, its underlying mechanism in maize with a defensive function to Exserohilum turcicum (E. turcicum) remains poorly understood. Here, we isolated and characterized a novel ERF transcription factor, designated ZmERF061, from maize. Phylogenetic analysis revealed that ZmERF061 is a member of B3 group in the ERF family. qRT-PCR assays showed that the expression of ZmERF061 is significantly induced by E. turcicum inoculation and hormone treatments with salicylic acid (SA) and methyl jasmonate (MeJA). ZmERF061 was proved to function as a nucleus-localized transcription activator and specifically bind to the GCC-box element. zmerf061 mutant lines resulted in enhanced susceptibility to E. turcicum via decreasing the expression of ZmPR10.1 and ZmPR10.2 and the activity of antioxidant defense system. zmerf061 mutant lines increased the expression of the SA signaling-related gene ZmPR1a and decreased the expression of the jasmonic acid (JA) signaling-related gene ZmLox1 after infection with E. turcicum. In addition, ZmERF061 could interact with ZmMPK6-1. These results suggested that ZmERF061 plays an important role in response to E. turcicum and may be useful in genetic engineering breeding.

Genome-wide identification and functional analysis of the ERF2 gene family in response to disease resistance against Stemphylium lycopersici in tomato

BackgroundAPETALA2/ethylene responsive factor (AP2/ERF) transcription factors are a plant-specific family of transcription factors and one of the largest families of transcription factors. Ethylene response factors (ERF) regulate plant growth, development, and responses to biotic and abiotic stress. In a previous study, the ERF2 gene was significantly upregulated in both resistant and susceptible tomato cultivars in response to Stemphylium lycopersici. The main purpose of this study was to systematically analyze the ERF family and to explore the mechanism of ERF2 in tomato plants resisting pathogen infection by the Virus-induced Gene Silencing technique.ResultsIn this experiment, 134 ERF genes were explored and subjected to bioinformatic analysis and divided into twelve groups. The spatiotemporal expression characteristics of ERF transcription factor gene family in tomato were diverse. Combined with RNA-seq, we found that the expression of 18 ERF transcription factors increased after inoculation with S. lycopersici. In ERF2-silenced plants, the susceptible phenotype was observed after inoculation with S. lycopersici. The hypersensitive response and ROS production were decreased in the ERF2-silenced plants. Physiological analyses showed that the superoxide dismutase, peroxidase and catalase activities were lower in ERF2-silenced plants than in control plants, and the SA and JA contents were lower in ERF2-silenced plants than in control plants after inoculation with S. lycopersici. Furthermore, the results indicated that ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance.ConclusionsIn this study, we identified and analyzed members of the tomato ERF family by bioinformatics methods and classified, described and analyzed these genes. Subsequently, we used VIGS technology to significantly reduce the expression of ERF2 in tomatoes. The results showed that ERF2 had a positive effect on tomato resistance to S. lycopersici. Interestingly, ERF2 played a key role in multiple SA, JA and ROS signaling pathways to confer resistance to invasion by S. lycopersici. In addition, ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance to S. lycopersici. In summary, this study provides gene resources for breeding for disease resistance in tomato.

Ustilago maydis effector Jsi1 interacts with Topless corepressor, hijacking plant jasmonate/ethylene signaling.

Summary Ustilago maydis is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins that suppress immune responses and redirect the host metabolism in favor of the pathogen. As effectors play a critical role during plant colonization, their identification and functional characterization are essential to understanding biotrophy and disease.Using biochemical, molecular, and transcriptomic techniques, we performed a functional characterization of the U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1).Jsi1 interacts with several members of the plant corepressor family Topless/Topless related (TPL/TPR). Jsi1 expression in Zea mays and Arabidopsis thaliana leads to transcriptional induction of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signaling pathway. In A. thaliana, activation of the ERF branch leads to biotrophic susceptibility. Jsi1 likely activates the ERF branch via an EAR (ET‐responsive element binding‐factor‐associated amphiphilic repression) motif, which resembles EAR motifs from plant ERF transcription factors, that interacts with TPL/TPR proteins.EAR‐motif‐containing effector candidates were identified from different fungal species, including Magnaporthe oryzae, Sporisorium scitamineum, and Sporisorium reilianum. Interaction between plant TPL proteins and these effector candidates from biotrophic and hemibiotrophic fungi indicates the convergent evolution of effectors modulating the TPL/TPR corepressor hub.

A Novel ERF Transcription Factor, ZmERF105, Positively Regulates Maize Resistance to Exserohilum turcicum.

The ethylene response factor (ERF) plays a crucial role in plant innate immunity. However, the molecular function of ERF in response to Exserohilum turcicum (E. turcicum) remains unknown in maize. In this study, a novel ERF gene, designated as ZmERF105, was firstly isolated and characterized. The ZmERF105 protein contains an APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) domain and a conserved LSPLSPHP motif in its C-terminal region. ZmERF105 protein was exclusively localized to the nucleus. ZmERF105 expression responded to E. turcicum treatment. Yeast one-hybrid and transcription activity assays revealed that ZmERF105 is an activator of transcription and binds to GCC-box elements. Over-expression of ZmERF105 was shown to increase maize resistance against E. turcicum, and erf105 mutant lines displayed opposite phenotype. Moreover, the activities of superoxide dismutase (SOD) and peroxidase (POD) in the ZmERF105 over-expression lines were markedly higher than in the wild-type maize lines (WT) after infection with E. turcicum, and were compromised in the erf105 mutant lines. Simultaneously, ZmERF105 over-expression lines enhanced the expression of several pathogenesis-related (PR) genes, including ZmPR1a, ZmPR2, ZmPR5, ZmPR10.1, and ZmPR10.2 after infection with E. turcicum. In contrast, the expression of PR genes was reduced in erf105 mutant lines. Our work reveals that ZmERF105 as a novel player of the ERF network and positively regulates the maize resistance response to E. turcicum.

Transcriptome Analysis Identifies Two Ethylene Response Factors That Regulate Proanthocyanidin Biosynthesis During Malus Crabapple Fruit Development.

Proanthocyanidins (PAs) are a class of flavonoid compounds in plants that play many important roles in pest and disease resistance and are beneficial components of the human diet. The crabapple (Malus) provides an excellent model to study PA biosynthesis and metabolism; therefore, to gain insights into the PA regulatory network in Malus plants, we performed RNA-seq profiling of fruits of the ‘Flame’ cultivar at five sequential developmental stages. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that differentially expressed genes (DEGs) related to the functional category ‘plant hormone signal transduction’ were significantly enriched during fruit development. Further analysis showed that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ETHYLENE INSENSITIVE 3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis. Additionally, 12 DEGs, including 10 ERFs, 1 MYB, and 1 bHLH transcription factor, associated with PA biosynthesis were identified using WGCNA. The expression patterns of these genes correlated with PA accumulation trends and transcriptome data from qRT-PCR analysis. The expression of RAP2-4 (RELATED TO APETALA 2-4) and RAV1 (related to ABI3/VP1), which belong to the ERF transcription factor family, showed the greatest correlations with PAs accumulation among the 12 identified TFs. Agrobacterium mediated-transient overexpression of the RAP2-4 led to an increase in PA abundance in crabapple leaves and apple fruits, and the opposite results were observed in RAV1-overexpressed crabapple leaves and apple fruits. Moreover, a yeast one-hybrid assay showed that RAP2-4 and RAV1 specifically bound the promoters of the PA biosynthetic genes McLAR1 and McANR2, respectively. These results indicate that RAP2-4 act as an inducer and RAV1 act as a repressor of PA biosynthesis by regulating the expression of the PA biosynthetic genes McLAR1 and McANR2. Taken together, we identified two potential regulators of PA biosynthesis and provide new insights into the ethylene-PA regulatory network.

Overexpression of ethylene response factor ERF96 gene enhances selenium tolerance in Arabidopsis

Ethylene response factors (ERFs) are involved in the regulation of plant responses to biotic and abiotic stresses. Here we provide evidence for a role of ERF96, a member of the ERF transcription factor group IX, in selenite tolerance in Arabidopsis. ERF96 gene was rapidly up-regulated in response to selenite stress. Overexpression of ERF96 enhanced Arabidopsis resistance to selenite stress, while ERF96-silenced plants demonstrated wild-type (WT) resistance to selenite. In addition, the overexpression plants had significantly lower selenium (Se) content in shoots when subjected to selenite stress. Further investigation indicated that overexpression of ERF96 reduced transcript levels of selenite/phosphate transporters PHT1;1 and PHT2;1, which influenced Arabidopsis Se uptake and allocation in the presence of selenite. Moreover, our experiments showed that overexpression of ERF96 enhanced Arabidopsis antioxidant activity. Under selenite stress, ERF96-overexpressing lines exhibited the significant increases in catalase (CAT) and glutathione peroxidase (GPX) activities as well as the glutathione (GSH) content, while had a decrease in reactive oxygen species (ROS) accumulation compared to WT. Taken together, our results demonstrate that ERF96 plays a positive role in the regulation of selenite tolerance in Arabidopsis.

The ethylene responsive factor CdERF1 from bermudagrass (Cynodon dactylon) positively regulates cold tolerance

Cold stress is one of the major environmental factors that limit growth and utilization of bermudagrass [Cynodon dactylon (L.) Pers], a prominent warm-season turfgrass. However, the molecular mechanism of cold response in bermudagrass remains largely unknown. In this study, we characterized a cold-responsive ERF (ethylene responsive factor) transcription factor, CdERF1, from bermudagrass. CdERF1 expression was induced by cold, drought and salinity stresses. The CdERF1 protein was nucleus-localized and encompassed transcriptional activation activity. Transgenic Arabidopsis plants overexpressing CdERF1 showed enhanced cold tolerance, whereas CdERF1-underexpressing bermudagrass plants via virus induced gene silencing (VIGS) method exhibited reduced cold resistance compared with control, respectively. Under cold stress, electrolyte leakage (EL), malondialdehyde (MDA), H2O2 and O2− contents were reduced, while the activities of SOD and POD were elevated in transgenic Arabidopsis. By contrast, these above physiological indicators in CdERF1-underexpressing bermudagrass exhibited the opposite trend. To further explore the possible molecular mechanism of bermudagrass cold stress response, the RNA-Seq analyses were performed. The result indicated that overexpression of CdERF1 activated a subset of stress-related genes in transgenic Arabidopsis, such as CBF2, pEARLI1 (lipid transfer protein), PER71 (peroxidase) and LTP (lipid transfer protein). Interestingly, under-expression of CdERF1 suppressed the transcription of many genes in CdERF1-underexpressing bermudagrass, also including pEARLI1 (lipid transfer protein) and PER70 (peroxidase). All these results revealed that CdERF1 positively regulates plant cold response probably by activating stress-related genes, PODs, CBF2 and LTPs. This study also suggests that CdERF1 may be an ideal candidate in the effort to improve cold tolerance of bermudagrass in the further molecular breeding.

Expression Analysis of ERFs during Storage under Modified Atmosphere Packaging (High-concentration of CO2) of Fresh-cut Lotus Root

Ethylene response factor (ERF) genes have been characterized in numerous plants, where they are associated with responses to biotic and abiotic stress. Modified atmosphere packaging (MAP) is an effective treatment to prevent lotus root browning. However, the possible relationship between ERF transcription factors and lotus root browning under MAP remains unexplored. In this study, the effects of phenol, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) enzyme activities; and PPO, PAL, POD, and ERF gene expression on fresh-cut lotus root browning were studied with MAP. The expression pattern of ERF2/5 correlated highly with the degree of browning. It is suggested that NnERF2/5 can be used as an important candidate gene for the regulation of fresh-cut lotus root browning under MAP, and the correlation of each gene should be studied further.

An ERF transcription factor enhances plant resistance to Myzus persicae and Spodoptera litura

Aphids are sap-sucking insects and are pests that cause substantial damage to many crops worldwide. Spodoptera litura is a leaf-chewing insect and poses a severe threat to many species of vegetable and cotton plants. In this paper, we studied an ethylene-responsive factor (ERF) that enhances plant resistance to sap-sucking and leaf-chewing insects. ERF11-like was obtained from Chinese cabbage via reverse transcription-polymerase chain reaction and was termed BrERF11b. The putative protein encoded by the BrERF11b gene includes 168 amino acid residues and contains an EAR motif. The plasmid pBI121-BrERF11b was constructed and transformed into tobacco plants (Nicotiana tabacum cv. K326 and N. benthamiana) by the Agrobacterium-mediated transformation method. The function of the BrERF11b gene in the resistance of the transgenic plants to Myzus persicae and S. litura was assayed. The results showed that the weights of the S. litura larvae fed the transgenic plant line were significantly reduced from 28 to 76% compared to those of the S. litura larvae fed the control plants (wild type), and the transgenic plant line KLine12 significantly reduced the survival rate of S. litura by 62.5%. The colony growth of the apteral aphids fed the transgenic plant line was significantly reduced from 22.5 to 63.4% compared to that of the apteral aphids fed the control plants. Compared to control plants, the transgenic plant line significantly reduced the survival rate of the aphids from 46 to 55%. These results first indicate that the BrERF11b gene significantly enhances plant resistance to S. litura and M. persicae.

BcERF070, a novel ERF (ethylene-response factor) transcription factor from non-heading Chinese cabbage, affects the accumulation of ascorbic acid by regulating ascorbic acid-related genes

L-Ascorbic acid (AsA) is an important nutrient in non-heading Chinese cabbage (Brassica rapa ssp. chinensis). To understand the regulatory mechanism of AsA accumulation, we crossed “Wutacai” and “Erqing” to obtain F2 plants and then constructed two pools (AsA-High and AsA-Low) of F2-generation plants to further analyze the differentially expressed genes (DEGs) by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technology. Among the DEGs, BcERF070 showed a significantly higher expression level in “Wutacai” and F2 plants with high AsA than in “Erqing” and F2 plants with low AsA, suggesting that BcERF070 is involved in the accumulation of AsA. To further understand the function of BcERF070 in AsA accumulation, we obtained BcERF070-silenced plants and BcERF070-overexpressed Arabidopsis thaliana plants. It was found that the downregulation of BcERF070 resulted in a decrease in AsA content, and the upregulation of BcERF070 resulted in an increase in AsA content. To evaluate the mechanism of BcERF070 involvement in the accumulation of AsA, the leaves of BcERF070-silenced and control plants were subjected to transcriptome sequencing. Twelve DEGs including BrAPXs were found to be associated with ascorbic acid. The qRT-PCR analysis on BcERF070-silenced plants and BcERF070-overexpressed plants further demonstrated the reliability of the transcriptome sequencing. Finally, based on analysis of the cis-acting elements of the promoter of 12 AsA-related genes, we speculated that BcERF070 may act on the DRE (dehydration-responsive element) of seven target gene promoters to regulate AsA content. In conclusion, a novel transcription factor, BcERF070, was revealed in this study, which affected the accumulation of AsA by regulating genes involved in the ascorbate biosynthesis and metabolic pathway. This information will help in understanding the regulatory mechanism of BcERF070 involvement in the accumulation of AsA in plants.

Apple ethylene response factor MdERF11 confers resistance to fungal pathogen Botryosphaeria dothidea

Ethylene response factor (ERF) is a plant-specific transcription factor involved in many biological processes including root formation, hypocotyl elongation, fruit ripening, organ senescence and stress responses, as well as fruit quality formation. However, its underlying mechanism in plant pathogen defense against Botryosphaeria dothidea (B. dothidea) remains poorly understood. Here, we isolate MdERF11, an apple nucleus-localized ERF transcription factor, from apple cultivar ‘Royal Gala’. qRT-PCR assays show that the expression of MdERF11 is significantly induced in apple fruits after B. dothidea infection. Overexpression of MdERF11 gene in apple calli significantly increases the resistance to B.dothidea infection, while silencing MdERF11 in apple calli results in reduced resistance. Ectopic expression of MdERF11 in Arabidopsis also exhibits enhanced resistance to B. dothidea infection compared to that of wild type. Infections in apple calli and Arabidopsis leaves by B. dothidea respectively cause an increase in endogenous levels of salicylic acid (SA) followed by induction of SA synthesis-related and signaling-related gene expression. Taken together, these findings illustrate a potential mechanism by which MdERF11 elevates plant pathogen defense against B. dothidea by regulating SA synthesis pathway.

The ERF transcription factor MdERF38 promotes drought stress-induced anthocyanin biosynthesis in apple.

Drought stress induces anthocyanin biosynthesis in many plant species, but the underlying molecular mechanism remains unclear. Ethylene response factors (ERFs) play key roles in plant growth and various stress responses, including affecting anthocyanin biosynthesis. Here, we characterized an ERF protein, MdERF38, which is involved in drought stress-induced anthocyanin biosynthesis. Biochemical and molecular analyses showed that MdERF38 interacted with MdMYB1, a positive modulator of anthocyanin biosynthesis, and facilitated the binding of MdMYB1 to its target genes. Therefore, MdERF38 promoted anthocyanin biosynthesis in response to drought stress. Furthermore, we found that MdBT2, a negative modulator of anthocyanin biosynthesis, decreased MdERF38-promoted anthocyanin biosynthesis by accelerating the degradation of the MdERF38 protein. In summary, our data provide a mechanism for drought stress-induced anthocyanin biosynthesis that involves dynamic modulation of MdERF38 at both transcriptional and post-translational levels.