Ethylene-responsive Transcription Factor Gene Research Articles

월동 배추의 추위 스트레스 반응성 ERF 유전자 발굴과 활용

To mitigate plant freeze damage caused by extreme weather events, we need to elucidate the freeze resistance mechanism of freeze-tolerant plants through molecular biological approaches. Our research focuses on identifying the cold stress responsive, ERF(Ethylene-Responsive transcription Factor) genes that confer freeze tolerance in overwintering Chinese cabbage. Additionally, we aim to explore the molecular basis for conferring freezing tolerance to other organisms through genetic transformation. Expression of the ERF, cold stress responsive gene in overwintering Chinese cabbage, leads to the synthesis of AFP(Antifreeze Protein), which results in cellular freezing tolerance. We identified and sequenced functionally important ERF domain regions within the ERF genes, which we named BrERF1 and BrERF2. Their responsiveness to cold stress was confirmed through RT-PCR and qRT-PCR. Furthermore, we propose strategies to introduce BrERF1 and BrERF2 insertion plasmids into other organisms for transformation. Significant improvements in the freezing tolerance of transgenic organisms are expected to improve the productivity of these target organisms. In particular, the development of permanently transgenic plants using the BrERFs gene offers the possibility of increasing plant production under cold stress.

GmERF54, an ERF Transcription Factor, Negatively Regulates the Resistance of Soybean to the Common Cutworm (Spodoptera litura Fabricius)

Soybean is attacked by various herbivorous insect pest species during the whole course of its life cycle in the field. It is important for soybean production to improve insect resistance by identifying and utilizing soybean endogenous insect-resistant genes. The ethylene-responsive transcription factor (ERF) plays a significant role in plant biotic and abiotic stresses; however, few studies focus on its role in insect resistance in soybean. Here, based on our previous common cutworm (CCW)-induced soybean transcriptome data, a soybean ERF gene GmERF54 was cloned, which responded to CCW feeding. Transcriptional analysis revealed that GmERF54 was ubiquitous in all soybean tissues and was expressed differently in insect-resistant and insect-susceptible soybean cultivars. RNA interference of GmERF54 increased the resistance to CCW, while the overexpression of GmERF54 decreased the resistance to CCW in transgenic soybean hairy roots compared with their controls. GmERF54 was localized to the nucleus, had transcriptional activation activity, and interacted with AP2/ERF GmPLT2. Several putative hormone response elements were predicted in the promoter sequence of GmERF54. Four putative elements were only found in the GmERF54 promoter sequence of insect-resistant cultivar Wanxianbaidongdou (WX), but not in the insect-susceptible cultivar Nannong 99-10 (99-10). GmERF54 promoter sequences of WX and 99-10 were cloned into the pCAMBIA1381z vector containing the β-glucuronidase (GUS) gene to generate GmERF54WX:GUS and GmERF5499-10:GUS recombinant vectors, respectively. GUS staining of soybean hairy roots containing GmERF54WX:GUS and GmERF5499-10:GUS showed that GmERF54 was induced by CCW attack and both MeJA (methyl jasmonate) and IAA (indole-3-acetic acid) treatments. Alleles in insect-resistant and insect-sensitive cultivars responded to these inductions differently. Overall, our results reveal that GmERF54 may be involved in the regulation of soybean resistance to CCW.

The submergence tolerance regulator SUB1A differentially coordinates molecular adaptation to submergence in mature and growing leaves of rice (Oryza sativa L.).

A typical adaptive response to submergence regulated by SUB1A, the ethylene-responsive transcription factor gene, is the restricted elongation of the uppermost leaves. However, the molecular and physiological functions of SUB1A have been characterized using entire shoot tissues, most of which are mature leaves that do not elongate under submergence. We aimed to identify leaf-type-specific and overlapping adaptations coordinated in SUB1A-dependent and -independent manners. To this end, we compared the transcriptomic and hormonal responses to submergence between mature and growing leaves using rice genotypes with and without SUB1A. Monosaccharide, branched-chain amino acid, and nucleoside metabolism, associated with ATP synthesis, were commonly activated in both leaf types regardless of genotype. In both leaf types, pathways involved in carbohydrate and nitrogen metabolism were suppressed by SUB1A, with more severe restriction in growing leaves that have a greater energy demand if SUB1A is absent. In growing leaves, accumulation of and responsiveness to growth-regulating hormones were properly modulated by SUB1A, which correlated with restricted elongation. In mature leaves, submergence-induced auxin accumulation was suppressed by SUB1A. This study demonstrates that different sets of hormonal pathways, both of which are modulated by SUB1A, contribute to distinct adaptive responses to submergence in mature and growing rice leaves.

A Large-Scale Genome-Wide Association Analyses of Ethiopian Sorghum Landrace Collection Reveal Loci Associated With Important Traits.

The eastern Africa region, Ethiopia and its surroundings, is considered as the center of origin and diversity for sorghum, and has contributed to global sorghum genetic improvement. The germplasm from this region harbors enormous genetic variation for various traits but little is known regarding the genetic architecture of most traits. Here, 1425 Ethiopian landrace accessions were phenotyped under field conditions for presence or absence of awns, panicle compactness and shape, panicle exsertion, pericarp color, glume cover, plant height and smut resistance under diverse environmental conditions in Ethiopia. In addition, F1 hybrids obtained from a subset of 1341 accessions crossed to an A1 cytoplasmic male sterile line, ATx623, were scored for fertility/sterility reactions. Subsequently, genotyping-by-sequencing generated a total of 879,407 SNPs from which 72,190 robust SNP markers were selected after stringent quality control (QC). Pairwise distance-based hierarchical clustering identified 11 distinct groups. Of the genotypes assigned to either one of the 11 sub-populations, 65% had high ancestry membership coefficient with the likelihood of more than 0.60 and the remaining 35% represented highly admixed accessions. A genome-wide association study (GWAS) identified loci and SNPs associated with aforementioned traits. GWAS based on compressed mixed linear model (CMLM) identified SNPs with significant association (FDR ≤ 0.05) to the different traits studied. The percentage of total phenotypic variation explained with significant SNPs across traits ranged from 2 to 43%. Candidate genes showing significant association with different traits were identified. The sorghum bHLH transcription factor, ABORTED MICROSPORES was identified as a strong candidate gene conditioning male fertility. Notably, sorghum CLAVATA1 receptor like kinase, known for regulation of plant growth, and the ETHYLENE RESPONSIVE TRANSCRIPTION FACTOR gene RAP2-7, known to suppress transition to flowering, were significantly associated with plant height. In addition, the YELLOW SEED1 like MYB transcription factor and TANNIN1 showed strong association with pericarp color validating previous observations. Overall, the genetic architecture of natural variation representing the complex Ethiopian sorghum germplasm was established. The study contributes to the characterization of genes and alleles controlling agronomic traits, and will serve as a source of markers for molecular breeding.

LcMKK, a MAPK kinase from Lycium chinense, confers cadmium tolerance in transgenic tobacco by transcriptional upregulation of ethylene responsive transcription factor gene.

Cadmium (Cd) is a highly toxic element to plants. Ethylene is an important phytohormone in the regulation of plant growth, development and stress response. Mitogen-activated protein kinase (MAPK) activation has been observed in plants exposed to Cd stress and was suggested to be involved in ethylene biosynthesis. We hypothesized that there may be a link between MAPK cascades and ethylene signalling in Cd-stressed plants. To test this hypothesis, the expression of LcMKK, LchERF and LcGSH1 genes, endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Our results showed that LcMKK gene expression can be induced by the treatment of Cd in L. chinense. The transgenic tobacco expressing 35S::LcMKK showed greater tolerance to Cd stress and enhanced expression of NtERF and NtGSH1 genes, indicating that LcMKK is associated with the enhanced expression level of ERF and GSH synthesis-related genes in tobacco. We also found that endogenous ethylene and GSH content can be induced by Cd stress in L. chinense, and inhibited by cotreatment with PD98059, an inhibitor of MAPK kinase. Evidences presented here suggest that under Cd stress, GSH accumulation occurred at least partially by enhanced LcMKK gene expression and the ethylene signal transduction pathways might be involved in this accumulation.

The glutathione synthesis may be regulated by cadmium-induced endogenous ethylene in Lycium chinense, and overexpression of an ethylene responsive transcription factor gene enhances tolerance to cadmium stress in tobacco

Glutathione (GSH) plays a pivotal role in heavy metal detoxification. Ethylene is one of the important plant hormones, which plays a critical role in triggering plant responses to different stresses such as cadmium (Cd) stress. Ethylene responsive transcription factor (ERF) belongs to one of the largest plant transcription factor families. ERF is known to be induced by ethylene and thus regulate multiple stress responses through the activation of stress-related genes. Until now, little has been done to explore the relationship among the accumulation of endogenous ethylene, ERF transcript levels and the GSH content in plants under Cd treatment and we will investigate these link. In this study, the gene transcript level of LchERF, LchGSH1 (gene responsible for the first-step GSH biosynthesis) and LchGSHS (gene responsible for the second-step GSH biosynthesis), endogenous ethylene accumulation, GSH content and Cd concentration in Lycium chinense with or without Cd stress treatment were studied. Furthermore, the transgenic tobacco expressing 35S::LchERF which grown under Cd stress condition was also investigated in this study. Our results showed that endogenous ethylene, LchERF, LchGSH1 and LchGSHS gene expression and GSH content can be induced by Cd treatment in L. chinense, however, reduced by co-treatment with 2-aminoethoxyvinlglycine (AVG), an inhibitor of ethylene biosynthesis. The transgenic tobacco expressing 35S::LchERF showed greater tolerance to Cd stress than non-transgenic plants. The expression of NtGSH1 and NtGSHS genes was increased in transgenic tobacco plants compared with non-transgenic plants, indicating that LchERF is associated with the expression level of GSH synthesis related genes in tobacco. Evidence was presented here that under Cd stress, GSH accumulation occurred at least partially via enhanced LchERF gene expression and the ethylene signal transduction pathways might be involved in this accumulation.

Transcriptomic analysis reveals ethylene as stimulator and auxin as regulator of adventitious root formation in petunia cuttings.

Adventitious root (AR) formation in the stem base (SB) of cuttings is the basis for propagation of many plant species and petunia is used as model to study this developmental process. Following AR formation from 2 to 192 hours post-excision (hpe) of cuttings, transcriptome analysis by microarray revealed a change of the character of the rooting zone from SB to root identity. The greatest shift in the number of differentially expressed genes was observed between 24 and 72 hpe, when the categories storage, mineral nutrient acquisition, anti-oxidative and secondary metabolism, and biotic stimuli showed a notable high number of induced genes. Analyses of phytohormone-related genes disclosed multifaceted changes of the auxin transport system, auxin conjugation and the auxin signal perception machinery indicating a reduction in auxin sensitivity and phase-specific responses of particular auxin-regulated genes. Genes involved in ethylene biosynthesis and action showed a more uniform pattern as a high number of respective genes were generally induced during the whole process of AR formation. The important role of ethylene for stimulating AR formation was demonstrated by the application of inhibitors of ethylene biosynthesis and perception as well as of the precursor aminocyclopropane-1-carboxylic acid, all changing the number and length of AR. A model is proposed showing the putative role of polar auxin transport and resulting auxin accumulation in initiation of subsequent changes in auxin homeostasis and signal perception with a particular role of Aux/IAA expression. These changes might in turn guide the entrance into the different phases of AR formation. Ethylene biosynthesis, which is stimulated by wounding and does probably also respond to other stresses and auxin, acts as important stimulator of AR formation probably via the expression of ethylene responsive transcription factor genes, whereas the timing of different phases seems to be controlled by auxin.

Increase in pectin deposition by overexpression of an ERF gene in cultured cells of Arabidopsis thaliana

Ethylene-responsive transcription factor (ERF) family genes, which are involved in regulation of metabolic pathways and/or are useful for metabolic engineering, were investigated in the cultured cells of Arabidopsis thaliana. The pectin content in the gelatinous precipitates after the ethanol precipitation of extracts derived from calli of a transgenic cell line, A17, overexpressing an ERF gene (At1g44830), increased in comparison with the control. Expression of genes involved in pectin biosynthesis was up-regulated in the A17 calli. Overexpression of the ERF gene coordinately activates the pectin biosynthetic pathway genes and increases the content of pectin. These results therefore will be useful as a genetic resource for engineering pectin biosynthesis in plants.

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

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