Ethylene Response Factor Subfamily Research Articles

GhERF.B4-15D: A Member of ERF Subfamily B4 Group Positively Regulates the Resistance against Verticillium dahliae in Upland Cotton.

Verticillium wilt is a fungal disease in upland cotton and exerts a significant effect on growth and potential productivity. This disease is mainly caused by V. dahliae Kleb. Ethylene response factor (ERF) is one of the superfamilies of transcription factors that is involved in the development and environmental adaption of crops. A total of 30 ERF.B4 group members were detected in upland cotton and divided into 6 subgroups. Gene structures, conserved motifs, and domain analysis revealed that members in each subgroup are highly conserved. Further, the 30 GhERF.B4 group members were distributed on 18 chromosomes, and 36 gene synteny relationships were found among them. GhERF.B4 genes were ubiquitously expressed in various tissues and developmental stages of cotton. Amongst them, GhERF.B4-15D was predominantly expressed in roots, and its expression was induced by V. dahliae infection. In addition, GhERF.B4-15D responded to methyl jasmonate (MeJA), methyl salicylate (MeSA), and ethylene (ET) phytohormones. It was also found that the V. dahliae resistance was enhanced due to overexpression of GhERF.B4-15D in Arabidopsis thaliana. On the contrary, interference of GhERF.B4-15D by virus-induced gene silencing (VIGS) technology decreased the V. dahliae resistance level in upland cotton. The subcellular localization experiment showed that GhERF.B4-15D was located in the nucleus. Yeast two-hybrid (Y2H) and luciferase complementation (LUC) approaches demonstrated that GhERF.B4-15D interacted with GhDREB1B. Additionally, the V. dahliae resistance was significantly decreased in GhDREB1B knockdowns. Our results showed that GhERF.B4-15D plays a role during V. dahliae infection in cotton.

Genome-wide identification and expression analysis of the Pisum sativum (L.) APETALA2/ethylene-responsive factor (AP2/ERF) gene family reveals functions in drought and cold stresses.

AP2/ERF (APETALA2/Ethylene Response Factor) is a family of transcription factors that play essential roles in regulating gene expression in response to various environmental stimuli, including biotic and abiotic stresses, hormone signaling, and developmental processes. Pisum sativum (L.), commonly known as garden pea, is a winter crop sensitive to high temperatures and can also be affected by extreme cold and drought conditions. This study performed a genome-wide analysis of AP2/ERF genes and identified 153 AP2/ERF genes in P. sativum. Based on the conserved AP2/ERF domain and sequence homology, they were classified into AP2 (APETALA2), ERF (Ethylene Response Factor), DREB (Dehydration responsive element-binding), RAV (Related to Abscisic Acid Insensitive 3/ Viviparous 1) and Soloist subfamily. The DREB and ERF subfamily were further divided into groups A1-6 and B1-B6. Tandem and segmental duplication events were more frequent in the ERF subfamily, which can have important implications for their evolution and functional diversification. Under cold stress, the expression of DREB1A was highly induced in leaves, whereas DREB1B was suppressed. Similarly, the DREB2A, DREB2C, DREB2E, and DREB2F were induced in leaves under drought stress. The putative target genes of AP2/ERF transcription factors are highly diversified, suggesting that they play essential roles in various physiological responses in plants, including responses to biotic and abiotic stresses as well as developmental processes. Thus, this study of AP2/ERF genes and their functions provides valuable insight into how P. sativum responds to different environmental conditions, including cold and drought stresses.

ERF subfamily transcription factors and their function in plant responses to abiotic stresses.

Ethylene Responsive Factor (ERF) subfamily comprise the largest number of proteins in the plant AP2/ERF superfamily, and have been most extensively studied on the biological functions. Members of this subfamily have been proven to regulate plant resistances to various abiotic stresses, such as drought, salinity, chilling and some other adversities. Under these stresses, ERFs are usually activated by mitogen-activated protein kinase induced phosphorylation or escape from ubiquitin-ligase enzymes, and then form complex with nucleic proteins before binding to cis-element in promoter regions of stress responsive genes. In this review, we will discuss the phylogenetic relationships among the ERF subfamily proteins, summarize molecular mechanism how the transcriptional activity of ERFs been regulated and how ERFs of different subgroup regulate the transcription of stress responsive genes, such as high-affinity K+ transporter gene PalHKT1;2, reactive oxygen species related genes LcLTP, LcPrx, and LcRP, flavonoids synthesis related genes FtF3H and LhMYBSPLATTER, etc. Though increasing researches demonstrate that ERFs are involved in various abiotic stresses, very few interact proteins and target genes of them have been comprehensively annotated. Hence, future research prospects are described on the mechanisms of how stress signals been transited to ERFs and how ERFs regulate the transcriptional expression of stress responsive genes.

Identification of AP2/ERF transcription factors in Tetrastigma hemsleyanum revealed the specific roles of ERF46 under cold stress.

Tetrastigma hemsleyanum (T. hemsleyanum) is a traditional medicinal plant that is widely used in China. Cultivated T. hemsleyanum usually encounters cold stress, limiting its growth and quality at key developmental stages. APETALA2 (AP2)/ethylene-responsive factor (ERF) transcription factors (TFs) comprise one of the largest gene superfamilies in plants and are widely involved in biotic and abiotic stresses. To reveal the roles of AP2/ERF TFs during T. hemsleyanum development, 70 AP2/ERF TFs were identified in T. hemsleyanum. Among them, 18 and 2 TFs were classified into the AP2 and RAV families, respectively. The other 50 TFs belonged to the ERF family and were further divided into the ERF and (dehydration reaction element binding factor) DREB subfamilies. The ERF subfamily contained 46 TFs, while the DREB subfamily contained 4 TFs. Phylogenetic analysis indicated that AP2/ERF TFs could be classified into five groups, in which 10 conserved motifs were confirmed. Several motifs were group- or subgroup-specific, implying that they were significant for the functions of the AP2/ERF TFs of these clades. In addition, 70 AP2/ERF TFs from the five groups were used for an expression pattern analysis under three low-temperature levels, namely, –4, 0, and 4°C. The majority of these AP2/ERF TFs exhibited a positive response to cold stress conditions. Specifically, ThERF5, ThERF31, ThERF46, and ThERF55 demonstrated a more sensitive response to cold stress. Moreover, AP2/ERF TFs exhibited specific expression patterns under cold stress. Transient overexpression and RNA interference indicated that ThERF46 has a specific tolerance to cold stress. These new insights provide the basis for further studies on the roles of AP2/ERF TFs in cold stress tolerance in T. hemsleyanum.

Identification and Characterization of the ERF Subfamily B3 Group Revealed GhERF13.12 Improves Salt Tolerance in Upland Cotton.

The APETALA2 (AP2)/ethylene response factor plays vital functions in response to environmental stimulus. The ethylene response factor (ERF) subfamily B3 group belongs to the AP2/ERF superfamily and contains a single AP2/ERF domain. Phylogenetic analysis of the ERF subfamily B3 group genes from Arabdiposis thaliana, Gossypium arboreum, Gossypium hirsutum, and Gossypium raimondii made it possible to divide them into three groups and showed that the ERF subfamily B3 group genes are conserved in cotton. Collinearity analysis identified172 orthologous/paralogous gene pairs between G. arboreum and G. hirsutum; 178 between G. hirsutum and G. raimondii; and 1,392 in G. hirsutum. The GhERF subfamily B3 group gene family experienced massive gene family expansion through either segmental or whole genome duplication events, with most genes showing signature compatible with the action of purifying selection during evolution. Most G. hirsutum ERF subfamily B3 group genes are responsive to salt stress. GhERF13.12 transgenic Arabidopsis showed enhanced salt stress tolerance and exhibited regulation of related biochemical parameters and enhanced expression of genes participating in ABA signaling, proline biosynthesis, and ROS scavenging. In addition, the silencing of the GhERF13.12 gene leads to increased sensitivity to salt stress in cotton. These results indicate that the ERF subfamily B3 group had remained conserved during evolution and that GhERF13.12 induces salt stress tolerance in Arabidopsis and cotton.

Molecular analysis of ERF subfamily genes during coffee somatic embryogenesis

With ample cultivation and economic potential, coffee is one of the main commodities in the world. Despite its vast cultivation, there remain problems related to its productivity as a consequence of pests and diseases, questions regarding its physiology, and difficulty with its propagation. Due to such challenges, the application of biotechnological tools, such as tissue culture and molecular markers, has become essential in the search for improvements to coffee cultivation. Among tissue culture techniques, somatic embryogenesis is considered the best form of micropropagation in vitro since it is possible to produce plants that are identical to the parent plant. It is therefore essential to identify the genes involved in the process of somatic embryogenesis, as well as to understand their functions. Different studies have shown the expression of ethylene response factor (ERF) genes in different tissues and under different conditions, mainly in response to biotic and abiotic stresses. However, little is known about their role during somatic embryogenesis. Thus, this study is aimed to identify and analyze the expression patterns of ERF subfamily genes during Coffea arabica L. somatic embryogenesis. The EST-contig6, 9, and 27 were confirmed to be the ethylene response factor 8, 13, and 12 genes from Coffea arabica L., respectively, and their relative expression analysis suggests that they may be involved in coffee somatic embryogenesis. The expression patterns of ethylene response factor 8 and 13 in embryogenic calluses and embryogenic cell suspensions showed that these genes can potentially act as somatic embryogenic markers.

Genome-wide identification and expression profiles of ERF subfamily transcription factors in Zea mays.

The Ethylene-Response Factor (ERF) subfamily transcription factors (TFs) belong to the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily and play a vital role in plant growth and development. However, identification and analysis of the ERF subfamily genes in maize have not yet been performed at genome-wide level. In this study, a total of 76 ERF subfamily TFs were identified and were found to be unevenly distributed on the maize chromosomes. These maize ERF (ZmERF) TFs were classified into six groups, namely groups B1 to B6, based on phylogenetic analysis. Synteny analysis showed that 50, 54, and 58 of the ZmERF genes were orthologous to those in rice, Brachypodium, and Sorghum, respectively. Cis-element analysis showed that elements related to plant growth and development, hormones, and abiotic stress were identified in the promoter region of ZmERF genes. Expression profiles suggested that ZmERF genes might participate in plant development and in response to salinity and drought stresses. Our findings lay a foundation and provide clues for understanding the biological functions of ERF TFs in maize.

Molecular cloning and expression analysis of three ThERFs involved in the response to waterlogging stress of Taxodium 'Zhongshanshan406', and subcellular localization of the gene products.

As a subfamily of the APETALA 2/ethylene response element binding protein (AP2/EREBP) transcription factor superfamily, the ethylene response factor (ERF) is widely involved in the regulation of growth and response to various abiotic stresses in plants, and has been shown to be the main transcription factor regulating transcription of the genes related to hypoxia and waterlogging stress. In this study, three ThERF genes, with significant differences in expression profile in response to flooding stress, were identified from the transcriptomics data acquired from Taxodium hybrid ‘Zhongshanshan 406’ (T. mucronatum Tenore × T. distichum (L.) Rich) under waterlogging stress: ThERF15, ThERF39 and ThRAP2.3 (GenBank ID: KY463467, KY463468 and KY463470, respectively).The full-length cDNA of each of the three ERFs was obtained using the RACE (rapid amplification cDNA ends) method, and all three were intron-free. Multiple protein sequence alignments indicated that ThERF15, ThERF39 and ThRAP2.3 proteins all had only one AP2-ERF domain and belonged to the ERF subfamily. A transient gene expression assay demonstrated that ThERF15, ThERF39 and ThRAP2.3 were all localized to the nucleus. Real-time quantitative PCR (qPCR) revealed that the expression of ThERF15, ThERF39 and ThRAP2.3 exhibited significant differences, compared with the control, in response to two levels of flooding treatment (half-flooding or total-submergence) of ‘Zhongshanshan 406’. Quantification of ethylene concentration revealed that ethylene was more relevant to the level of expression than the period of flooding treatment. Based on the experimental results above, ThERF15, ThERF39 and ThRAP2.3 were identified as being related to the regulation of downstream flooding- responsive gene expression in ‘Zhongshanshan 406’. ThRAP2.3 is most likely to be a key downstream-response ERF gene to respond to the output of the ethylene signal generated by flooding stress.

Expansion and stress responses of the AP2/EREBP superfamily in cotton

BackgroundThe allotetraploid cotton originated from one hybridization event between an extant progenitor of Gosssypium herbaceum (A1) or G. arboreum (A2) and another progenitor, G. raimondii Ulbrich (D5) 1–1.5 million years ago (Mya). The APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factors constitute one of the largest and most conserved gene families in plants. They are characterized by their AP2 domain, which comprises 60–70 amino acids, and are classified into four main subfamilies: the APETALA2 (AP2), Related to ABI3/VP1 (RAV), Dehydration-Responsive Element Binding protein (DREB) and Ethylene-Responsive Factor (ERF) subfamilies. The AP2/EREBP genes play crucial roles in plant growth, development and biotic and abiotic stress responses. Hence, understanding the molecular characteristics of cotton stress tolerance and gene family expansion would undoubtedly facilitate cotton resistance breeding and evolution research.ResultsA total of 269 AP2/EREBP genes were identified in the G. raimondii (D5) cotton genome. The protein domain architecture and intron/exon structure are simple and relatively conserved within each subfamily. They are distributed throughout all chromosomes but are clustered on various chromosomes due to genomic tandem duplication. We identified 73 tandem duplicated genes and 221 segmental duplicated gene pairs which contributed to the expansion of AP2/EREBP superfamily. Of them, tandem duplication was the most important force of the expansion of the B3 group. Transcriptome analysis showed that 504 AP2/EREBP genes were expressed in at least one tested G. hirsutum TM-1 tissues. In G. hirsutum, 151 non-repeated genes of the DREB and ERF subfamily genes were responsive to different stresses: 132 genes were induced by cold, 63 genes by drought and 94 genes by heat. qRT-PCR confirmed that 13 GhDREB and 15 GhERF genes were induced by cold and/or drought. No transcripts detected for 53 of the 111 tandem duplicated genes in TM-1. In addition, some homoeologous genes showed biased expression toward either A-or D-subgenome.ConclusionsThe AP2/EREBP genes were obviously expanded in Gossypium. The GhDREB and GhERF genes play crucial roles in cotton stress responses. Our genome-wide analysis of AP2/EREBP genes in cotton provides valuable information for characterizing the molecular functions of AP2/EREBP genes and reveals insights into their evolution in polyploid plants.

Structure analysis and expression pattern of the ERF transcription factor family in poplar

The ethylene response factor (ERF) family is one of the largest families of plant-specific transcription factors, and plays significant roles in plant growth and development, and stress tolerance processes. To understand the different structure and expression pattern of the ERF family, a total of 209 poplar ERF transcription factor genes were identified in silico. Based on the alignment of AP2/ERF domains, 209 PtERF proteins were classified into ten phylogenetic groups (I–X), and amino acid residues Gly-5 (G), Arg-7 (R), Glu-17 (E), Trp-36 (W), Leu-37 (L), Gly-38 (G), and Ala-46 (A) are conserved among these proteins. Protein structure analysis revealed that all poplar ERF family members contained the WLG motif (motif-1), but motif-6 was mainly conserved in the ERF subfamily. In addition, 209 genes were classified into three clusters under the salt stress condition: the lowly expressed Cluster (L), intermediately expressed Cluster (M1 and M2), and highly expressed Cluster (H1 and H2), in which some genes from highly expressed cluster were involved in salt stress response. Moreover, quantitative RT-PCR was used to analyze the tissue-specific expression pattern of 71 highly expressed genes. All of these results are of interest to better understand how and where each ERF functions in poplar.

The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.

RAP2.6L mRNA Level in Oil Palm (Elaeis guineensis) Leaf, Inflorescence Explants and Calli Produced From Them

The RAP2.6L is a member of the ERF (ethylene response factor) subfamily β‐4 of the ERF/APETALA2 transcription factor gene family. These preliminary studies seek to investigate the mRNA level of RAP2.6L gene in oil palm (Elaeis guineensis) leaf, inflorescence explants and the calli produced from them, with a view to understanding the molecular basis of direct and indirect organogenesis. Callus was induced and proliferated from non‐chlorophyllous leaf and immature inflorescence tissues using Murashige and Skoog medium. This was supplemented with different concentrations of auxins: 2,4‐D and NAA, ranging from 100‐120 mg/l and 170‐200 mg/l respectively. The times taken for callus induction, percentage callusing explants and weight of fresh callus after 20 weeks of culture initiation were recorded. Callus production was seen to vary in time of initial response and quantity produced for both 2,4‐D and NAA treatments. The leaf tissues yielded more callus in less time than the inflorescence tissues. Treatment 100mg/l 2,4‐D was effective in producing callus in 11 % of leaf explants, while 185 mg/l NAA was more effective in producing callus in 8 % of same. The level of RAP2.6L mRNA was measured with reverse transcription polymerase chain reaction (RT‐PCR). The results obtained indicated that there was no significant difference in the RAP2.6L mRNA level among the explants and associated calli, suggesting that both explants were equally amenable to indirect organogenesis.

Genome-Wide Identification and Phylogenetic Analysis of the ERF Gene Family in Melon

Members of the ERF transcription factor family have significant functions in regulating gene expression in response to biotic and abiotic stresses. In melon (Cucumis melo L.), however, few ERF genes have been studied. In this study, we performed a comprehensive computational analysis of 119 ERF family genes and identified 136 ERF superfamily genes in melon. We present a complete overview of ERF family gene in melon, including the phylogeny, gene structures, and putative conserved motifs of melon ERF family proteins, as well as a comparative analysis between ERF subfamily genes in cucumber. The AP2/ERF gene superfamily in melon can be classified into three families, ERF (ethylene-responsive factor), RAV (related to ABI3/VP1), and AP2 (APETALA2), which comprise 119, 4, and 13 members, respectively. The ERF family in melon was divided into 11 groups, designated I to XI, as in tomato. Our motif analysis indicated that most conserved motifs outside of the AP2/ERF domain are distributed between different clades in the phylogenetic tree. Most ERF family genes have no introns. Based on the observation that 11 of these groups existed in melon, we concluded that the chief functional diversification in the ERF family predated the divergence between monocots and dicots. In our analysis of expression patterns, ERF subfamily genes were distributed widely throughout melon plant tissues and most of the ERF subfamily genes were detected in all seven types of tissues. Some ERF subfamily genes show expression patterns characteristic of tissue-specific expression, implying their probability of performing various roles in growth and development in melon.

Isolation and identification of an AP2/ERF factor that binds an allelic cis-element of rice gene LRK6

Allelic expression of the rice yield-related gene, leucine-rich receptor-like kinase 6 (LRK6), in the hybrid of 93-11 (Oryza sativa L. subsp. Indica var. 93-11) and Nipponbare (O. sativa L. subsp. Japonica var. Nipponbare) is determined by allelic promoter cis-elements. Using deletion analysis of the LRK6 promoter, we identified two distinct regions that might contribute to LRK6 expression. Sequence alignment revealed differences in these LRK6 promoter regions in 93-11 and Nipponbare. One of the segments, named differential sequence of LRK6 promoter 2 (DSLP2), contains potential transcription factor binding sites. Using a yeast one-hybrid assay, we isolated an ethylene-responsive factor (ERF) protein that binds to DSLP2. Sequence analysis and a GCC-box assay showed that the ERF gene, O. sativa ERF 3 (OsERF3), which belongs to ERF subfamily class II, has a conserved ERF domain and an ERF-associated amphiphilic repression repressor motif. We used an in vivo mutation assay to identify a new motif (5'-TAA(A)GT-3') located in DSLP2, which interacts with OsERF3. These results suggest that OsERF3, an AP2 (APETALA 2 Gene)/ERF transcription factor, binds the LRK6 promoter at this new motif, which might cause differential expression of LRK6 in the 93-11/Nipponbare hybrid.

Cloning and Expression Profile of an ERF Gene Isolated from Cold-Stressed Poplar Cells (Populus nigra)

We analyzed a gene in the ethylene responsive factor (ERF) family, which plays an important role in allowing plants to respond to various environmental cues, isolated from suspension cultured cells of Populus nigra subjected to cold stress. A comparison of gene homology with transcription factors of the AP2/ERF family revealed a well conserved AP2/ERF domain in the predicted amino acid sequence of PncERF1. However, it has no consensus sequences around the AP2/ERF domain, which is a characteristic sequence of the CBF/DREB subfamily belonging to the ERF family. PncERF1 likely belongs to the ERF subfamily, which is the other ERF subfamily. Expression analysis showed no expression of PncERF1 at room temperature, at which cells grew normally, although it was expressed following cold treatment for 24 h. Therefore, PncERF1 was predicted to play a role in poplar cell survival in cold environments.

Expression Profiling of Cultivar-related Genes in Satsuma Mandarins, Miyagawa Wase and Ueno Wase

Satsuma mandarin (Citrus unshiu Marc.) is characterized by its tender peel and seedless nature. The representative cultivar of Satsuma mandarin is Miyagawa wase. Another cultivar, Ueno wase was developed from the bud mutation of a Miyagawa wase tree. Compared to Miyagawa wase fruit, Ueno wase fruit is known to have less albedo, higher sugar content, and to mature earlier. To find genes related to this bud mutation, a citrus 300 k DNA microarray was developed for gene expression analyses of the fruits and leaves of each cultivar. When up-regulated genes were classified in the Cluster of Orthologous Groups of proteins database, the number of genes included differed significantly between the two cultivars. Ten genes, including cytochrome P450, α-mannosidase, S-adenosylmethionine decarboxylase, carboxyesterase, anthranilate synthase, glycosyl hydrolase family-17, ethylene response factor subfamily, and alcohol dehydrogenase, showing significant differences in gene expression by DNA microarray were selected, and their expression patterns were examined by RT-PCR. The information gained from genes that are up (or down)-regulated upon bud mutation may explain the observed phenotypes, while also increasing the understanding of genes related to citrus bud mutation.

Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.

Tobacco (Nicotiana tabacum) synthesizes nicotine and related pyridine alkaloids in the root, and their synthesis increases upon herbivory on the leaf via a jasmonate-mediated signaling cascade. Regulatory NIC loci that positively regulate nicotine biosynthesis have been genetically identified, and their mutant alleles have been used to breed low-nicotine tobacco varieties. Here, we report that the NIC2 locus, originally called locus B, comprises clustered transcription factor genes of an ethylene response factor (ERF) subfamily; in the nic2 mutant, at least seven ERF genes are deleted altogether. Overexpression, suppression, and dominant repression experiments using transgenic tobacco roots showed both functional redundancy and divergence among the NIC2-locus ERF genes. These transcription factors recognized a GCC-box element in the promoter of a nicotine pathway gene and specifically activated all known structural genes in the pathway. The NIC2-locus ERF genes are expressed in the root and upregulated by jasmonate with kinetics that are distinct among the members. Thus, gene duplication events generated a cluster of highly homologous transcription factor genes with transcriptional and functional diversity. The NIC2-locus ERFs are close homologs of ORCA3, a jasmonate-responsive transcriptional activator of indole alkaloid biosynthesis in Catharanthus roseus, indicating that the NIC2/ORCA3 ERF subfamily was recruited independently to regulate jasmonate-inducible secondary metabolism in distinct plant lineages.

Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco

A new member of the AP2/ERF transcription factor family, GmERF3, was isolated from soybean. Sequence analysis showed that GmERF3 contained an AP2/ERF domain of 58 amino acids and two putative nuclear localization signal (NLS) domains. It belonged to a group IV protein in the ERF (ethylene response factor) subfamily as typified by a conserved N-terminal motif [MCGGAI(I/L)]. Expression of GmERF3 was induced by treatments with high salinity, drought, abscisic acid (ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and soybean mosaic virus (SMV), whereas there was no significant GmERF3 mRNA accumulation under cold stress treatment. GmERF3 could bind to the GCC box and DRE/CRT element, and was targeted to the nucleus when transiently expressed in onion epidermal cells. The GmERF3 protein fused to the GAL4 DNA-binding domain to activate transcription of reporter genes in yeast. Ectopic expression of the GmERF3 gene in transgenic tobacco plants induced the expression of some PR genes and enhanced resistance against infection by Ralstonia solanacearum, Alternaria alternata, and tobacco mosaic virus (TMV), and gave tolerance to high salinity and dehydration stresses. Furthermore, overexpression of GmERF3 in transgenic tobacco led to higher levels of free proline and soluble carbohydrates compared to wild-type plants under drought conditions. The overall results suggested that GmERF3 as an AP2/ERF transcription factor may play dual roles in response to biotic and abiotic stresses in plants.

Gene Expression Programs during Shoot, Root, and Callus Development in Arabidopsis Tissue Culture

Shoots can be regenerated from Arabidopsis (Arabidopsis thaliana) root explants in tissue culture through a two-step process requiring preincubation on an auxin-rich callus induction medium. Regenerating tissues can be directed along different developmental pathways leading to the formation of shoots, new roots, or callus by transferring to the appropriate organ induction medium. Using gene-profiling methods, we identified groups of genes that serve as molecular signatures of the different developmental processes, i.e. genes that were specifically up- or down-regulated on one developmental pathway, but not on others. One transcription factor gene that was up-regulated during early shoot development was RAP2.6L (At5g13330), a member of the ERF (ethylene response factor) subfamily B-4 of the ERF/APETALA2 transcription factor gene family. RAP2.6L functions in shoot regeneration because T-DNA knockdown mutations in the gene reduced the efficiency of shoot formation in tissue culture, but not normal embryo or seedling development. RAP2.6L promoter:beta-glucuronidase fusions demonstrated that the up-regulation of the gene during shoot regeneration was, at least in part, transcriptionally controlled. The promoter:beta-glucuronidase fusions also demonstrated that RAP2.6L expression was localized to the shoot and emerging leaves, but expression declined in the leaf lamina as leaves expanded. T-DNA knockdown mutations in RAP2.6L reduced the expression of many genes that are normally up-regulated during shoot development including CUP-SHAPED COTYLEDON2 that is involved in shoot meristem specification. Thus, RAP2.6L appears to be part of a network involved in regulating the expression of many other genes in shoot regeneration.