ERF Gene Clusters: Working Together to Regulate Metabolism

Recent literature has aimed the spotlight at clusters of the jasmonate-response APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors (TFs) for their roles in regulating plant specialized metabolism. Clustered ERFs represent a subset of the large ERF TF family; the evolutionary and regulatory relationships in these transcriptional genomic neighborhoods are exciting topics of molecular biology.Homologous members of an ERF cluster share overlapping but distinct functions, forming a regulatory hub that fine-tunes a complex metabolic pathway.The distinct functions of individual ERFs within a cluster allow biotechnological exploration to reprogram metabolic pathways.Compared with clusters in other TF families [e.g., basic helix-loop-helix (bHLH) or MYB], ERF clusters have been more extensively studied and thus serve as a good model for the understanding of TF clusters. Plants produce structurally diverse specialized metabolites, including bioactive alkaloids and terpenoids, in response to biotic and abiotic environmental stresses. The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors (TFs) play key roles in regulating biosynthesis of specialized metabolites. Increasing genomic and functional evidence shows that a subset of the ERF genes occurs in clusters on the chromosomes. These jasmonate-responsive ERF TF gene clusters control the biosynthesis of many important metabolites, from natural products, such as nicotine and steroidal glycoalkaloids (SGAs), to pharmaceuticals, such as artemisinin, vinblastine, and vincristine. Here, we review the function, regulation, and evolution of ERF clusters and highlight recent advances in understanding the distinct roles of clustered ERF genes and their possible application in metabolic engineering. Plants produce structurally diverse specialized metabolites, including bioactive alkaloids and terpenoids, in response to biotic and abiotic environmental stresses. The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors (TFs) play key roles in regulating biosynthesis of specialized metabolites. Increasing genomic and functional evidence shows that a subset of the ERF genes occurs in clusters on the chromosomes. These jasmonate-responsive ERF TF gene clusters control the biosynthesis of many important metabolites, from natural products, such as nicotine and steroidal glycoalkaloids (SGAs), to pharmaceuticals, such as artemisinin, vinblastine, and vincristine. Here, we review the function, regulation, and evolution of ERF clusters and highlight recent advances in understanding the distinct roles of clustered ERF genes and their possible application in metabolic engineering. Plants synthesize and accumulate a diverse array of specialized metabolites (see Glossary), including bioactive alkaloids and terpenoids that confer adaptive advantages during growth in fluctuating environmental conditions. Humans have long been exploiting plant-derived metabolites, or phytochemicals, as traditional medicines, drugs, dyes, perfumes, and industrial feedstock. Well-known examples of plant-derived clinical drugs include the sesquiterpene lactone artemisinin, the diterpenoid taxol, and the monoterpenoid indole alkaloids (MIAs), vincristine and vinblastine [1.Scossa F. et al.The integration of metabolomics and next-generation sequencing data to elucidate the pathways of natural product metabolism in medicinal plants.Planta Med. 2018; 84: 855-873Crossref PubMed Scopus (29) Google Scholar]. Recent molecular, genomic, and bioinformatics approaches have facilitated the prediction and identification of a large number of genes that encode enzymes for the biosynthesis of specialized metabolites [2.Anarat-Cappillino G. Sattely S.E. The chemical logic of plant natural product biosynthesis.Curr. Opin. Plant Biol. 2014; 19: 51-58Crossref PubMed Scopus (36) Google Scholar,3.Moore B.M. et al.Robust prediction of specialized metabolism genes through machine learning.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 2344-2353Crossref PubMed Scopus (36) Google Scholar]. By comparison, the regulatory genes governing these metabolic enzymes are less well characterized. Metabolic genes associated with specialized pathways are dynamically regulated in response to developmental and environmental stimuli. This regulation generally occurs at the level of transcription [4.Colinas M. Goossens A. Combinatorial transcriptional control of plant specialized metabolism.Trends Plant Sci. 2018; 23: 324-336Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar], and the massive metabolic flow in plants requires the coordinated expression of multiple metabolic and transport genes. Transcription factors (TFs) often mediate such coordinated expression (Box 1) [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar]. For example, TFs in the MYB and basic helix-loop-helix (bHLH) families regulate the anthocyanin and related flavonoid biosynthetic pathways in a wide range of species [6.Chezem W.R. Clay N.K. Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs.Phytochemistry. 2016; 131: 26-43Crossref PubMed Scopus (74) Google Scholar]. Genetic variation in regulatory genes contributes to the vast diversity of these metabolites found in nature [7.Huang D. et al.Subfunctionalization of the Ruby2-Ruby1 gene cluster during the domestication of citrus.Nat. Plants. 2018; 4: 930-941Crossref PubMed Scopus (53) Google Scholar,8.Springer N. et al.Challenges of translating gene regulatory information into agronomic improvements.Trends Plant Sci. 2019; 24: 1075-1082Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]. Indeed, recent advances in understanding biosynthesis of colorless specialized metabolites further reinforce the importance of transcriptional regulation of diverse metabolic processes in plants [9.Shoji T. et al.Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.Plant Cell. 2010; 22: 3390-3409Crossref PubMed Scopus (164) Google Scholar, 10.Shang Y. et al.Biosynthesis, regulation, and domestication of bitterness in cucumber.Science. 2014; 28: 1084-1088Crossref Scopus (232) Google Scholar, 11.Shanchez-Perez R. et al.Mutation of a bHLH transcription factor allowed almond domestication.Science. 2019; 364: 1095-1098Crossref PubMed Scopus (54) Google Scholar].Box 1Have TFs Primed the Metabolic Evolution?In a TF regulon, one or a set of TFs regulate a group of noncontiguous genes, such as genes in a particular metabolic pathway. TFs and the associated regulatory networks undergo significant modifications over time, driving metabolic diversification. Because of the ability of a TF to recognize and control multiple gene promoters, it is often the case that a single TF regulates evolutionarily diverse but functionally linked metabolic pathways. This concept is consistent with the involvement of evolutionarily conserved TFs in the regulation of multiple specialized pathways. Catalytic innovations through mutational changes of metabolic enzymes are largely responsible for the increase in chemo-diversity in nature [67.Moghe G.D. Last R.L. Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism.Plant Physiol. 2015; 169: 1512-1523PubMed Google Scholar]. Nevertheless, there must be limits to the extent that an enzyme can be modified without affecting protein stability and function. A limited repertoire of enzymes has been exploited in a combinatorial manner (Figure I). The recruitment model for metabolic evolution [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar] proposes that TFs play a priming role in the process of combining enzymes into a pathway, wherein a series of metabolic genes are repeatedly recruited into regulons under the control of TFs, gaining cognate cis-regulatory elements (Figure I). In this model, the functional expression of metabolic enzymes primed by TFs would be followed by catalytic modification (not depicted in Figure I) [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar]. In a TF regulon, one or a set of TFs regulate a group of noncontiguous genes, such as genes in a particular metabolic pathway. TFs and the associated regulatory networks undergo significant modifications over time, driving metabolic diversification. Because of the ability of a TF to recognize and control multiple gene promoters, it is often the case that a single TF regulates evolutionarily diverse but functionally linked metabolic pathways. This concept is consistent with the involvement of evolutionarily conserved TFs in the regulation of multiple specialized pathways. Catalytic innovations through mutational changes of metabolic enzymes are largely responsible for the increase in chemo-diversity in nature [67.Moghe G.D. Last R.L. Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism.Plant Physiol. 2015; 169: 1512-1523PubMed Google Scholar]. Nevertheless, there must be limits to the extent that an enzyme can be modified without affecting protein stability and function. A limited repertoire of enzymes has been exploited in a combinatorial manner (Figure I). The recruitment model for metabolic evolution [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar] proposes that TFs play a priming role in the process of combining enzymes into a pathway, wherein a series of metabolic genes are repeatedly recruited into regulons under the control of TFs, gaining cognate cis-regulatory elements (Figure I). In this model, the functional expression of metabolic enzymes primed by TFs would be followed by catalytic modification (not depicted in Figure I) [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar]. Jasmonates (JAs), a group of fatty acid-derived phytohormones, are key signals in plants, especially in defense responses to herbivores and necrotrophic pathogens [12.Wasternack C. Hause B. Jasmonatea: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany.Ann. Bot. 2013; 111: 1021-1058Crossref PubMed Scopus (1345) Google Scholar, 13.Goossens A. et al.Jasmontes: signal transduction components and their roles in environmental stress response.Plant Mol. Biol. 2016; 91: 673-689Crossref PubMed Scopus (109) Google Scholar, 14.Howe G.A. et al.Modularity in jasmonate signaling for multistress resilience.Annu. Rev. Plant Biol. 2018; 69: 387-415Crossref PubMed Scopus (207) Google Scholar]. JA treatment readily elicits the production of a large number of specialized metabolites that likely play adaptive roles in plant defense [15.Zhou M. Memelink J. Jasmonate-responsive transcription factors regulating plant secondary metabolism.Biotechnol. Adv. 2016; 34: 441-449Crossref PubMed Scopus (173) Google Scholar,16.Goossens J. et al.Role and functioning of bHLH transcription factors in jasmonate signalling.J. Exp. Bot. 2017; 68: 1333-1347PubMed Google Scholar]. JA signaling is mediated through JA-stimulated, proteasome-dependent degradation of JAZ repressor proteins and the resulting activation of TFs, such as the bHLH-family member MYC2 [13.Goossens A. et al.Jasmontes: signal transduction components and their roles in environmental stress response.Plant Mol. Biol. 2016; 91: 673-689Crossref PubMed Scopus (109) Google Scholar, 14.Howe G.A. et al.Modularity in jasmonate signaling for multistress resilience.Annu. Rev. Plant Biol. 2018; 69: 387-415Crossref PubMed Scopus (207) Google Scholar, 15.Zhou M. Memelink J. Jasmonate-responsive transcription factors regulating plant secondary metabolism.Biotechnol. Adv. 2016; 34: 441-449Crossref PubMed Scopus (173) Google Scholar, 16.Goossens J. et al.Role and functioning of bHLH transcription factors in jasmonate signalling.J. Exp. Bot. 2017; 68: 1333-1347PubMed Google Scholar]. This central JA signaling cascade is further connected to downstream metabolic processes through other TFs [5.Shoji T. The recruitment model of metabolic evolution: jasmonate-responsive transcription factors and a conceptual model for the evolution of metabolic pathways.Front. Plant Sci. 2019; 10: 560Crossref PubMed Scopus (14) Google Scholar,15.Zhou M. Memelink J. Jasmonate-responsive transcription factors regulating plant secondary metabolism.Biotechnol. Adv. 2016; 34: 441-449Crossref PubMed Scopus (173) Google Scholar,16.Goossens J. et al.Role and functioning of bHLH transcription factors in jasmonate signalling.J. Exp. Bot. 2017; 68: 1333-1347PubMed Google Scholar], including the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of TFs. The plant-specific AP2/ERF family is one of the largest TF families, with typically >100 members in each species. AP2/ERF TFs are classified into four subfamilies, namely AP2, ERF, RAV, and Soloist, and are responsible for the regulation of a diverse range of developmental processes and defense mechanisms [17.Licausi F. et al.APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs.New Phytol. 2013; 199: 639-649Crossref PubMed Scopus (513) Google Scholar]. The characteristic DNA-binding domain of AP2/ERF TFs, which consists of a sheet with three β-strands followed by an α-helix, recognizes specific cis-regulatory elements, including the GCC-box element (5′-AGCCGCC-3′) [18.Allen M.D. et al.A novel mode of DNA recognition by a β-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA.EMBO J. 1998; : 5484-5496Crossref PubMed Scopus (371) Google Scholar]. In 2000, Van der Fits and Memelink discovered the Octadecanoid-derivative Responsive Catharanthus AP2-domain 3 (ORCA3) TF, which regulates MIA biosynthesis in Catharanthus roseus [19.Van der Fits I. Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism.Science. 2000; 289: 295-297Crossref PubMed Scopus (713) Google Scholar]. Since then, a small group of related ERF TFs in clade II of group IXa [9.Shoji T. et al.Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.Plant Cell. 2010; 22: 3390-3409Crossref PubMed Scopus (164) Google Scholar,20.Nakano T. et al.Genome-wide analysis of ERF gene family in Arabidopsis and rice.Plant Physiol. 2006; 140: 411-432Crossref PubMed Scopus (1337) Google Scholar,21.Shoji T. et al.Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.Plant Physiol. 2013; 162: 977-990Crossref PubMed Scopus (40) Google Scholar] has emerged as JA-responsive transcriptional regulators in a diverse set of specialized metabolite pathways from several plant lineages (Figure 1). These ERFs include ORCA2, ORCA3, ORCA4, ORCA5, and ORCA6, which regulate MIAs in C. roseus [19.Van der Fits I. Memelink J. ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism.Science. 2000; 289: 295-297Crossref PubMed Scopus (713) Google Scholar,22.Menke F.L. et al.A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.EMBO J. 1999; 18: 4455-4463Crossref PubMed Scopus (343) Google Scholar, 23.Li C.Y. et al.The ORCA2 transcription factor play a key role in regulation of the terpenoid indole alkaloid pathway.BMC Plant Biol. 2013; 13: 155Crossref PubMed Scopus (68) Google Scholar, 24.Paul P. et al.A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.New Phytol. 2017; 213: 1107-1123Crossref PubMed Scopus (69) Google Scholar, 25.Paul P. et al.Mutually regulated AP2/ERF gene clusters modulate biosynthesis of specialized metabolites in plants.Plant Physiol. 2020; 182: 840-856Crossref PubMed Scopus (24) Google Scholar, 26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar, 27.Colinas M. et al.A modular system regulates specialized metabolite pathway branch choice in the medicinal plant Catharanthus roseus.bioRxiv. 2020; (Published online May 5, 2020. https://doi.org/10.1101/2020.05.04.075671)Google Scholar], OpERF2, which regulates MIAs in Ophiorrhiza pumila [28.Udomsom N. et al.Function of AP2/ERF transcription factors involved in the regulation of specialized metabolism in Ophiorrhiza pumila revealed by transcriptomics and metabolomics.Front. Plant Sci. 2016; 7: 1861Crossref PubMed Scopus (30) Google Scholar], ERF189 and ORC1/ERF221, which regulate nicotine in tobacco (Nicotiana tabacum) [9.Shoji T. et al.Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.Plant Cell. 2010; 22: 3390-3409Crossref PubMed Scopus (164) Google Scholar,29.De Boer K. et al.APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis.Plant J. 2011; 66: 1053-1065Crossref PubMed Scopus (150) Google Scholar], JA-Responsive ERF4 (JRE4)/Glycoalkaoid Metabolism 9 (GAME9), which regulates steroidal glycoalkaloids (SGAs) in tomato (Solanum lycopersicum) and potato (Solanum tuberosum) [30.Cárdenas P.D. et al.GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.Nat. Commun. 2016; 7: 10657Crossref PubMed Scopus (141) Google Scholar, 31.Thagun C. et al.Jasmonate-responsive ERF transcription factors regulate steroidal glycoalkaloid biosynthesis in tomato.Plant Cell Physiol. 2016; 57: 961-975Crossref PubMed Scopus (65) Google Scholar, 32.Nakayasu M. et al.JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.Plant J. 2018; 94: 975-990Crossref PubMed Scopus (32) Google Scholar], and AaORA, which regulates artemisinin in Artemisia annua [33.Lu X. et al.AaORA, a trichome-specific AP2/ERF transcription factors, is a positive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytils cinerea.New Phytol. 2013; 198: 1191-1202Crossref PubMed Scopus (162) Google Scholar,34.Ma Y.N. et al.Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua.Sci. Adv. 2018; 4: 9357Crossref Scopus (38) Google Scholar]. JA-responsive ERFs differentially target cis-regulatory elements, including the canonical GCC-box and the related but distinct GC-rich elements [21.Shoji T. et al.Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.Plant Physiol. 2013; 162: 977-990Crossref PubMed Scopus (40) Google Scholar]. For example, ORCA3 and JRE4, but not ERF189, bind to a GCC-box element [21.Shoji T. et al.Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.Plant Physiol. 2013; 162: 977-990Crossref PubMed Scopus (40) Google Scholar]. A small number of amino acid residues within the DNA-binding domain are key determinants of these divergent binding specificities [21.Shoji T. et al.Divergent DNA-binding specificities of a group of ETHYLENE RESPONSE FACTOR transcription factors involved in plant defense.Plant Physiol. 2013; 162: 977-990Crossref PubMed Scopus (40) Google Scholar]. Notably, putative binding sites for JRE4 and ERF189 have been found to be enriched in the promoter regions of sets of downstream structural genes [31.Thagun C. et al.Jasmonate-responsive ERF transcription factors regulate steroidal glycoalkaloid biosynthesis in tomato.Plant Cell Physiol. 2016; 57: 961-975Crossref PubMed Scopus (65) Google Scholar,35.Kajikawa M. et al.Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco.Plant Physiol. 2017; 174: 999-1011Crossref PubMed Scopus (38) Google Scholar,36.Xu S. et al.Wild tobacco genomes reveal the evolution reveal the evolution of nicotine biosynthesis.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 6133-6138Crossref PubMed Scopus (75) Google Scholar]. Generation of cognate cis-regulatory elements in the gene promoters allows repeated recruitment of structural genes into regulons under the control of conserved TFs (Box 1). Despite differences in DNA-binding preferences, ERFs generally have functions that are conserved among homologs in different species. For instance, the ERF189-targeted promoter of a tobacco nicotine biosynthetic gene, qunolinate phosphoribosyl transferase 2 (QPT2), is active in transgenic tomato and is regulated in the same manner as in tobacco; its promoter activity is JA-responsive, cell type-specific, and depends on function of the endogenous JRE4 [37.Shoji T. Hashimoto T. Expression of a nicotine biosynthesis gene depends on JRE4 transcription factor in heterogeneous tomato.J. Plant Res. 2019; 132: 173-180Crossref PubMed Scopus (6) Google Scholar]. In addition, ERF189 and ORCA5 can each regulate the other's endogenous target structural genes, resulting in metabolite accumulation upon heterologous expression in C. roseus and tobacco, respectively [24.Paul P. et al.A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.New Phytol. 2017; 213: 1107-1123Crossref PubMed Scopus (69) Google Scholar]. These results underscore the notion that orthologous ERFs can be functionally equivalent and exchangeable. In a wide range of eudicots, the JA-responsive ERF genes are present in multiple copies and often clustered in the genome (Figure 2). Arabidopsis (Arabidopsis thaliana) ERF13 (AtERF13) is a notable exception that exists as a single gene copy. By contrast, tobacco has two clusters of ten and five ERF genes [35.Kajikawa M. et al.Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco.Plant Physiol. 2017; 174: 999-1011Crossref PubMed Scopus (38) Google Scholar], potato has a cluster of eight [30.Cárdenas P.D. et al.GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.Nat. Commun. 2016; 7: 10657Crossref PubMed Scopus (141) Google Scholar], tomato has a cluster of five [30.Cárdenas P.D. et al.GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.Nat. Commun. 2016; 7: 10657Crossref PubMed Scopus (141) Google Scholar,31.Thagun C. et al.Jasmonate-responsive ERF transcription factors regulate steroidal glycoalkaloid biosynthesis in tomato.Plant Cell Physiol. 2016; 57: 961-975Crossref PubMed Scopus (65) Google Scholar], C. roseus has a cluster of five [26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar], Calotropis gigantea has a cluster of four [26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar], and Gelsemium sempervirens has a cluster of four [26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar] (Figure 2). In addition to group IXa clade II ERFs, ERFs in other groups, such as the group III C-repeat Binding Factor genes, are also found in clusters in the genomes [38.Gilmour S.J. et al.Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.Plant J. 1998; 16: 433-442Crossref PubMed Google Scholar,39.Zhang X. et al.Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.Plant J. 2004; 39: 905-919Crossref PubMed Scopus (317) Google Scholar]. Unlike the metabolic gene clusters that include genes encoding various classes of metabolic enzymes [40.Nützmann H.W. et al.Plant metabolic clusters: from genetics to genomics.New Phytol. 2016; 211: 771-789Crossref PubMed Scopus (151) Google Scholar,41.Nützmann H.W. et al.Active and repressed biosynthetic gene clusters have spatially distinct chromosome states.Proc. Natl. Acad. 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A. 2020; 117: 13800-13809Crossref PubMed Scopus (21) Google Scholar], the TF clusters generally consist of genes of one TF family organized in tandem [11.Shanchez-Perez R. et al.Mutation of a bHLH transcription factor allowed almond domestication.Science. 2019; 364: 1095-1098Crossref PubMed Scopus (54) Google Scholar,26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar,35.Kajikawa M. et al.Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco.Plant Physiol. 2017; 174: 999-1011Crossref PubMed Scopus (38) Google Scholar,38.Gilmour S.J. et al.Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.Plant J. 1998; 16: 433-442Crossref PubMed Google Scholar,39.Zhang X. et al.Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.Plant J. 2004; 39: 905-919Crossref PubMed Scopus (317) Google Scholar,42.Zhang P. et al.A segmental gene duplication generated differentially expressed myb-homologous genes in maize.Plant Cell. 2000; 12: 2311-2322Crossref PubMed Scopus (93) Google Scholar]. Repeated gene duplication, which occurs mainly through unequal crossing-over events, may contribute to the formation of these clusters [43.Magadum S. et al.Gene duplication as a major force in evolution.J. Genet. 2013; 92: 155-161Crossref PubMed Scopus (185) Google Scholar]. In many plant families, the phylogenetic relationships of the clustered ERFs suggest the likelihood of multiple, independent emergences of the ERF clusters (Figure 2). Within the large ERF gene family, a small subset of the genes form clusters [26.Singh S.K. et al.Revisiting the ORCA gene cluster that regulates terpenoid indole alkaloid biosynthesis in Catharanthus roseus.Plant Sci. 2020; 293: 110408Crossref PubMed Scopus (18) Google Scholar,35.Kajikawa M. et al.Genomic insights into the evolution of the nicotine biosynthesis pathway in tobacco.Plant Physiol. 2017; 174: 999-1011Crossref PubMed Scopus (38) Google Scholar,38.Gilmour S.J. et al.Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression.Plant J. 1998; 16: 433-442Crossref PubMed Google Scholar,39.Zhang X. et al.Freezing-sensitive tomato has a functional CBF cold response pathway, but a CBF regulon that differs from that of freezing-tolerant Arabidopsis.Plant J. 2004; 39: 905-919Crossref PubMed Scopus (317) Google Scholar], raising questions about the biological and evolutionary significance of ERF clusters. As a result of functional differentiation among the duplicate genes, proteins encoded by the clustered ERFs usually have overlapping but distinct roles. In Solanaceae lineages, ERF189 and JRE4 play predominantly regulatory roles in the biosynthesis of nicotine in tobacco and SGA in tomato, respectively, whereas other members in the ERF cluster have either a subsidiary function or no function in the regulation of alkaloid biosynthesis. Loss of ERF189 or JRE4 function results in a dramatic reduction of alkaloid production [32.Nakayasu M. et al.JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato.Plant J. 2018; 94: 975-990Crossref PubMed Scopus (32) Google Scholar,44.Hayashi S. et al.Genetic manipulation of transcriptional regulators alters nicotine biosynthesis in tobacco.Plant Cell Physiol. 2020; 61: 1041-1053Crossref PubMed Scopus (15) Google Scholar]. In tomato, overexpression of JRE3 (Figure 2), the closest homolog of JRE4, does not induce SGA formation [45.Abdelkareem A. et al.Identification of genes regulated by jasmonate- and salt-inducible transcription factor JRE3 in tomato.Plant Biotechnol. 2019; 36: 29-37Crossref PubMed Scopus (7) Google Scholar]. In tobacco, nicotine biosynthesis is induced only moderately by overexpression of ERF genes other than ERF189 [9.Shoji T. et al.Clustered transcription factor genes regulate nicotine biosynthesis in tobacco.Plant Cell. 2010; 22: 3390-3409Crossref PubMed Scopus (164) Google Scholar,46.Shoji T. Hashimoto T. DNA-binding and transcriptional activation properties of tobacco NIC2-locus ERF189 and r