ETHYLENE RESPONSE FACTOR6, A Central Regulator of Plant Growth in Response to Stress.

ETHYLENE RESPONSE FACTOR6 (ERF6) has emerged as a central player in stress-induced plant growth inhibition. It orchestrates complex pathways that enable plants to acclimate and thrive in challenging environments. In response to various abiotic and biotic stresses, ERF6 is promptly activated through both ethylene-dependent and -independent pathways, and contributes to enhanced stress tolerance mechanisms by activating a broad spectrum of genes at various developmental stages. Despite the crucial role of ERF6, there is currently a lack of published comprehensive insights into its function in plant growth and stress response. In this respect, based on the tight connection between ethylene and ERF6, we review the latest research findings on how ethylene regulates stress responses and the mechanisms involved. In addition, we summarize the trends and advances in ERF6-mediated plant performance under optimal and stressful conditions. Finally, we also highlight key questions and suggest potential paths to unravel the ERF6 regulon in future research.

Comprehensive transcriptome analysis of AP2/ERFs in Osmanthus fragrans reveals the role of OfERF017-mediated organic acid metabolism pathway in flower senescence.

Osmanthus fragrans is an ethylene-sensitive flower, and flower senescence was mediated by ethylene-responsive transcription factors (OfERFs). A total of 227 OfERFs were identified from O. fragrans, which were classified into five subfamilies: AP2 (35), DREB (57), ERF (125), RAV (6), and Soloist (4). Gene composition and structural analysis indicate that members of different subfamilies have different gene structures and conserved domains. Their gene promoter contains various functional responsive elements, including auxin, jasmonic acid, and other responsive elements. Among them, 124 OfAP2/ERF genes have expressed at any stage of flowering, and 10 of them may play roles in flowering or senescence. By comparative transcriptome analysis, OfAP2/ERFs affected by ethephon (ETH) and 5'-azacytidine (Aza) treatment were divided into three categories, which have various target gene sets. Importantly, these target gene sets participate in similar or different biological processes and metabolic pathways, suggesting that ethylene and DNA hypomethylation have crosstalk and a unique mechanism in regulating the flower senescence of O. fragrans. Co-expression analysis revealed that several key OfAP2/ERFs played a central role in organic acid metabolism and biosynthesis of branched-chain amino acids (BcAAs), among which OfERF017 was selected for further functional analysis. Overexpression of OfERF017 leads to significant enrichment of genes in organic acid metabolism pathways, which leads to a decrease in organic acid levels and promoting the flower senescence of O. fragrans. Together, these results give insights into the characteristics and functional analysis of OfAP2/ERF genes in O. fragrans.

Genome-wide analysis of the AP2/ERF gene family in Pennisetum glaucum and the negative role of PgRAV_01 in drought tolerance.

APETALA2/ethylene-responsive (AP2/ERF) plays crucial roles in resisting diverse stresses and in regulating plant growth and development. However, little is known regarding the structure and function of the AP2/ERF genes in pearl millet (Pennisetum glaucum). The AP2/ERF gene family may be involved in the development and maintenance of P. glaucum resilience to abiotic stresses, central to its role as a vital forage and cereal crop. In this study, PgAP2/ERF family members were identified and comprehensive bioinformatics analyses were performed, including determination of phylogenetic relationships, gene structures, conserved motifs, chromosomal localization, gene duplication, expression pattern, protein interaction network, and functional characterization of PgRAV_01 (Related to ABI3/VP1). In total, 78 PgAP2/ERF members were identified in the P. glaucum genome and classified into five subfamilies: AP2, ERF, DREB, RAV, and soloist. Members within the same clade of the PgAP2/ERF family showed similar gene structures and motif compositions. Six duplication events were identified in the PgAP2/ERF family; calculation of Ka/Ks values showed that purification selection dominated the evolution of PgAP2/ERFs. Subsequently, a potential interaction network of PgAP2/ERFs was generated to predict the interaction relationships. Additionally, abiotic stress expression analysis showed that most PgAP2/ERFs were induced in response to drought and heat stresses. Furthermore, overexpression of PgRAV_01 negatively regulated drought tolerance in Nicotiana benthamiana by reducing its antioxidant capacity and osmotic adjustment. Taken together, these results provide valuable insights into the characteristics and functions of PgAP2/ERF genes, with implications for abiotic stress tolerance, and will ultimately contribute to the genetic improvement of cereal crop breeding.

Genome-wide analysis of the ERF Family in Stephania japonica provides insights into the regulatory role in Cepharanthine biosynthesis.

Introduction: Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA) extracted from Stephania japonica, has received significant attention for its anti-coronavirus properties. While ethylene response factors (ERFs) have been reported to regulate the biosynthesis of various alkaloids, their role in regulating CEP biosynthesis remains unexplored. Methods: Genome-wide analysis of the ERF genes was performed with bioinformatics technology, and the expression patterns of different tissues, were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. The nuclear-localized ERF gene cluster was shown to directly bind to the promoters of several CEP-associated genes, as demonstrated by yeast one-hybrid assays and subcellular localization assays. Results: In this work, 59 SjERF genes were identified in the S. japonica genome and further categorized into ten subfamilies. Notably, a SjERF gene cluster containing three SjERF genes was found on chromosome 2. Yeast one-hybrid assays confirmed that the SjERF gene cluster can directly bind to the promoters of several CEP-associated genes, suggesting their crucial role in CEP metabolism. The SjERFs cluster-YFP fusion proteins were observed exclusively in the nuclei of Nicotiana benthamiana leaves. Tissue expression profiling revealed that 13 SjERFs exhibit high expression levels in the root, and the qRT-PCR results of six SjERFs were consistent with the RNA-Seq data. Furthermore, a co-expression network analysis demonstrated that 24 SjERFs were highly positively correlated with the contents of various alkaloids and expression levels of CEP biosynthetic genes. Conclusion: This study provides the first systematic identification and analysis of ERF transcription factors in the S.japonica genome, laying the foundation for the future functional research of SjERFs transcription factors.

Modulation of Plant Transcription Factors and Priming of Stress Tolerance by Plant Growth-Promoting Bacteria: A Systematic Review.

BACKGROUND: Plant growth-promoting bacteria (PGPB) have been shown to improve plant growth and stress tolerance through mechanisms including improved access to nutrients and biotic competition with pathogens. As such, the use of PGPB can help to address challenges to crop productivity, however, information on interactions between PGPB and their plant hosts, especially at the level of gene regulation, is distributed across diverse studies involving several different plants and PGPB. SCOPE: For this review, we analysed recent research publications reporting specifically on plant transcription factor (TF) expression in association with PGPB, to determine if there are any common findings and to identify gaps that offer opportunities for focused future research. CONCLUSIONS: The inoculation of plants with PGPB elicits a dynamic and temporal response. Initially, there is an upregulation of defence-responsive TFs, followed by their downregulation in an intermediate phase, and finally, another upregulation, providing longer term stress tolerance. PGPB-priming activates plant defences in the form of induced systemic resistance (ISR), often via the MAMP/MAPK pathways and involving one or more of the major plant hormone-signalling pathways and their crosstalk. Following PGPB-priming, the TFs families most commonly reported as expressed across different plants and for different pathogens are ERF and WRKY, while the TFs most commonly expressed across different plants for different abiotic stresses are ERF and DREB. There were inconsistencies between studies regarding the timing of the shift from the initial phase to the intermediate phase, and some of the TFs expressed during this process have not been fully characterized. This calls for more research to investigate the regulatory functions and phases of TF expression, to enhance crop resilience. Most reports on abiotic stresses have focused on salinity and drought, with fewer studies addressing nutrient deficiency, heavy metals, flooding, and other stresses, highlighting the need for further research in these areas.

Transcriptome analysis reveals the potential mechanism of methyl jasmonate alleviated ripening disorder in mango fruit at low temperature.

High susceptibility of mangoes to low temperature leads to ripening failure that restricts the marketability of products. This study investigated the effect of methyl jasmonate (MeJA) on ripening disorder and mechanism involved in mangoes during refrigeration. Results showed that 50 µM MeJA ameliorated ripening disorder, as indicated by accelerated advancement of ripening-related parameters. Transcriptome analysis revealed that 17,414 significantly differentially expressed genes were mainly enriched in ethylene synthesis, cell wall degradation, starch degradation and sugar transport. Moreover, 8 AP2/ERF transcription factors and 12 ripening-related genes were characterized via qRT-PCR. Afterwards, through the analysis of transcription factor binding sites and cis-acting elements, a regulatory network of ERFs mediated alleviation of ripening disorder conferred by MeJA was constructed. Finally, the interactions between MiERFs and the promoters of target genes were verified by yeast one-hybrid assay. Our findings provide a theoretical basis for improving cold tolerance via counteracting ripening disorder in mangoes.

Master Regulatory Transcription Factors in ß-Aminobutyric Acid-Induced Resistance (BABA-IR): A Perspective on Phytohormone Biosynthesis and Signaling in Arabidopsis thaliana and Hordeum vulgare.

The endogenous stress metabolite ß-aminobutyric acid (BABA) primes plants for enhanced resistance against abiotic and biotic stress by activating a complex phytohormone signaling network that includes abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). In this study, through stringent filtering, we identify 14 master regulatory transcription factors (TFs) from the DOF, AHL, and ERF families that potentially regulate the biosynthesis and signaling of these phytohormones. Transcriptional analysis of BABA-treated Arabidopsis thaliana and Hordeum vulgare suggests that DOF family TFs play a crucial role in stress response regulation in both species. BABA treatment in A. thaliana upregulates the TFs MNB1A and PBF and enhances the expression of the genes ICS1, EDS5, and WIN3 in the SA biosynthesis pathway, potentially boosting NPR1 and PR1 in the SA signaling pathway. Conversely, in H. vulgare, the BABA-induced upregulation of TF DOF5.8 may negatively regulate SA biosynthesis by downregulating ICS1, EDS5, and PR1. Additionally, in A. thaliana, BABA triggers the expression of TF PBF, which may result in the decreased expression of MYC2, a key gene in JA signaling. In contrast, H. vulgare exhibits increased expression of ERF2 TF, which could positively regulate the JA biosynthesis genes LOX and Tify9, along with the COI1 and JAZ genes involved in the JA signaling pathway. These findings offer new perspectives on the transcriptional regulation of phytohormones during plant priming.

PfERF106, a novel key transcription factor regulating the biosynthesis of floral terpenoids in Primula forbesii Franch.

BACKGROUND: Flowers can be a source of essential oils used in the manufacture of substances with high economic value. The ethylene response factor (ERF) gene family plays a key role in regulating secondary metabolite biosynthesis in plants. However, until now, little has been known about the involvement of ERF transcription factors (TFs) in floral terpenoid biosynthesis. RESULTS: In this study, an aromatic plant, Primula forbesii Franch., was used as research material to explore the key regulatory effects of PfERF106 on the biosynthesis of terpenoids. PfERF106, which encodes an IXb group ERF transcription factor, exhibited a consistent expression trend in the flowers of P. forbesii and was transcriptionally induced by exogenous ethylene. Transient silencing of PfERF106 in P. forbesii significantly decreased the relative contents of key floral terpenes, including (z)-ß-ocimene, sabinene, ß-pinene, γ-terpinene, linalool, eremophilene, α-ionone, and α-terpineol. In contrast, constitutive overexpression of PfERF106 in transgenic tobacco significantly increased the relative contents of key floral terpenes, including cis-3-hexen-1-ol, linalool, caryophyllene, cembrene, and sclareol. RNA sequencing of petals of PfERF106-silenced plants and empty-vector control plants revealed 52,711 expressed unigenes and 9,060 differentially expressed genes (DEGs). KEGG annotation analysis revealed that the DEGs were enriched for involvement in secondary metabolic biosynthetic pathways, including monoterpene and diterpene synthesis. Notably, 10 downregulated DEGs were determined to be the downstream target genes of PfERF106 affecting the biosynthesis of terpenoids in P. forbesii. CONCLUSION: This study characterized the key positive regulatory effects of PfERF106 on the biosynthesis of terpenoids, indicating high-quality genetic resources for aroma improvement in P. forbesii. Thus, this study advances the artificial and precise directional regulation of metabolic engineering of aromatic substances.

Jasmonic acid plays an important role in mediating retrograde signaling under mitochondrial translational stress to balance plant growth and defense.

Proper mitochondrial function is crucial to plant growth and development. Inhibition of mitochondrial translation leads to mitochondrial proteotoxic stress, which triggers a protective transcriptional response that regulates nuclear gene expression, commonly referred to as the mitochondrial unfolded protein response (UPRmt). Although UPRmt has been extensively studied in yeast and mammals, very little is known about UPRmt in plants. Here, we show that mitochondrial translational stress inhibits plant growth and development by inducing jasmonic acid (JA) biosynthesis and signaling. The inhibitory effect of mitochondrial translational stress on plant growth was alleviated in JA signaling defective mutants coi1-2, myc2, and myc234. Genetic analysis indicates that Arabidopsis mitochondrial ribosomal protein L1 (MRPL1), a key factor in UPRmt, regulates plant growth in a CORONATINE-INSENSITIVE1 (COI1)-dependent manner. Moreover, under mitochondrial translational stress, MYC2 showed direct binding to G-boxes in the ETHYLENE RESPONSE FACTOR 109 (ERF109) promoter. The induction of ERF109 expression enhances hydrogen peroxide (H2O2) production, which acts as a feedback loop to inhibit root growth. In addition, mutation of MRPL1 increases JA accumulation, reduces plant growth, and enhances biotic stress resistance. Overall, our findings reveal that JA plays an important role in mediating retrograde signaling under mitochondrial translational stress to balance plant growth and defense.

Genome-wide identification and comprehensive analysis of the AP2/ERF gene family in Prunus sibirica under low-temperature stress.

BACKGROUND: AP2/ERF transcription factors are involved in the regulation of growth, development, and stress response in plants. Although the gene family has been characterized in various species, such as Oryza sativa, Arabidopsis thaliana, and Populus trichocarpa, studies on the Prunus sibirica AP2/ERF (PsAP2/ERF) gene family are lacking. In this study, PsAP2/ERFs in P. sibirica were characterized by genomic and transcriptomic analyses. RESULTS: In the study, 112 PsAP2/ERFs were identified and categorized into 16 subfamilies. Within each subfamily, PsAP2/ERFs exhibited similar exon-intron structures and motif compositions. Additionally, 50 pairs of segmentally duplicated genes were identified within the PsAP2/ERF gene family. Our experimental results showed that 20 PsAP2/ERFs are highly expressed in leaves, roots, and pistils under low-temperature stress conditions. Among them, the expression of PsAP2/ERF21, PsAP2/ERF56 and PsAP2/ERF88 was significantly up-regulated during the treatment period, and it was hypothesised that members of the PsAP2/ERF family play an important role inlow temperature stress tolerance. CONCLUSIONS: This study improves our understanding of the molecular basis of development and low-temperature stress response in P. sibirica and provides a solid scientific foundation for further functional assays and evolutionary analyses of PsAP2/ERFs.

LkERF6 enhances drought and salt tolerance in transgenic tobacco by regulating ROS homeostasis.

The transcription factor Ethylene Responsive Factor (ERF) is crucial for responding to various environmental stressors. Proteins containing the ERF-associated amphiphilic repression (EAR) motif often inhibit gene expression. However, the functions of LkERF, an EAR motif-containing protein from Larix kaempferi, especially in reactive oxygen species (ROS) homeostasis, are not well understood. In the present research, we introduce a novel transcription factor, LkERF6, which contains an EAR motif and positively regulates gene expression, thereby enhancing drought and salt tolerance in tobacco. LkERF6 is classified within the ERF-B1 subfamily due to its conserved AP2/ERF domain and EAR motif. Subcellular localization assays demonstrated LkERF6 is primarily localized in the nucleus. Further analysis revealed that LkERF6 interacts with GCC and DRE elements and is significantly induced by NaCl and PEG6000. Moreover, LkERF6 transgenic tobacco plants exhibit lower ROS accumulation and higher levels of antioxidant enzyme activities. Additionally, correlation analysis identified a strong association between LkERF6 and three genes: LkSOD, LkCCS, and LkCAT. Y1H, EMAS, and DLR assays confirmed that LkERF6 directly interacts with the promoters of these genes through GCC-box and DRE-box to activate their expression. These findings shed new light on the function of EAR motif-containing transcription factors and highlight LkERF6's crucial role in enhancing abiotic stress resistance by activating multiple ROS clearance genes.

The abscisic acid-responsive transcriptional regulatory module CsERF110-CsERF53 orchestrates citrus fruit coloration.

Carotenoid biosynthesis is closely associated with abscisic acid (ABA) during the ripening process of non-climacteric fruits, but the regulatory mechanism that links ABA signaling to carotenoid metabolism remains largely unclear. Here, we identified two master regulators of ABA-mediated citrus fruit coloration, CsERF110 and CsERF53, which activate the expression of carotenoid metabolism genes (CsGGPPS, CsPSY, CsPDS, CsCRTISO, CsLCYB2, CsLCYE, CsHYD, CsZEP, and CsNCED2) to facilitate carotenoid accumulation. Further investigations showed that CsERF110 not only activates the expression of CsERF53 by binding to its promoter but also interacts with CsERF53 to form the transcriptional regulatory module CsERF110-CsERF53. We also discovered a positive feedback regulatory loop between the ABA signal and carotenoid metabolism regulated by the transcriptional regulatory module CsERF110-CsERF53. Our results reveal that the CsERF110-CsERF53 module responds to ABA signaling, thereby orchestrating citrus fruit coloration. Considering the importance of carotenoid content for citrus and many other carotenoid-rich crops, the revelation of molecular mechanisms that underlie ABA-mediated carotenoid biosynthesis in plants will facilitate the development of transgenic/gene-editing approaches, further contributing to improving the quality of citrus and other carotenoid-rich crops.

Genome-wide identification of the AP2/ERF gene family from Limonium bicolor and functional characterization of LbAP2/ERF32 under salt stress.

AP2/ERF transcription factors (TFs) play important roles in plant growth and development, plant morphogenesis and response to environmental stresses. However, their biological roles in recretohalophytes are still not fully revealed. Limonium bicolor L. is a typical recretohalophyte, which secretes excessive salt ions through the salt glands on the epidermis. Here, 64 LbAP2/ERF genes were identified in L. bicolor genome, which were unevenly distributed on the eight chromosomes. Cis-elements related to growth and development, stress response and phytohormone response are distributed in multiple LbAP2/ERF promoters. Expression analysis indicated that LbAP2/ERF genes responsed to NaCl, PEG and ABA. And the salt gland density, salt secretion of leaves and overall salt tolerance of LbAP2/ERF32 silenced lines were significantly reduced. In agreement, the genes related to salt gland development and ion transport were significantly changed in LbAP2/ERF32-silenced lines. Our findings provided fundamental information on the structure and evolutionary relationship of LbAP2/ERF gene family in salt gland development and salt secretion of L. bicolor and gave theoretical guideline for further functional study of LbAP2/ERF genes in response to abiotic stress.

Arabidopsis thaliana MYC2 and MYC3 Are Involved in Ethylene-Regulated Hypocotyl Growth as Negative Regulators.

The ethylene-regulated hypocotyl elongation of Arabidopsis thaliana involves many transcription factors. The specific role of MYC transcription factors in ethylene signal transduction is not completely understood. The results here revealed that two MYCs, MYC2 and MYC3, act as negative regulators in ethylene-suppressed hypocotyl elongation. Etiolated seedlings of the loss-of-function mutant of MYC2 or MYC3 were significantly longer than wild-type seedlings. Single- or double-null mutants of MYC2 and MYC3 displayed remarkably enhanced response to ACC(1-aminocyclopropane-1-carboxylate), the ethylene precursor, compared to wild-type seedlings. MYC2 and MYC3 directly bind to the promoter zone of ERF1, strongly suppressing its expression. Additionally, EIN3, a key component in ethylene signaling, interacts with MYC2 or MYC3 and significantly suppresses their binding to ERF1's promoter. MYC2 and MYC3 play crucial roles in the ethylene-regulated expression of functional genes. The results revealed the novel role and functional mechanism of these transcription factors in ethylene signal transduction. The findings provide valuable information for deepening our understanding of their role in regulating plant growth and responding to stress.

Auxin responsive factor MdARF17 promotes ethylene synthesis in apple fruits by activating MdERF003 expression.

KEY MESSAGE: Auxin (AUX) promotion of apple fruit ripening is ethylene-dependent, and AUX-MdARF17-MdERF003 plays a role in AUX-promoted ethylene synthesis in apple. Phytohormones play important roles in plant growth and fleshy fruit ripening, and the phytohormone auxin (AUX) can either promote or inhibit the ripening of fleshy fruits. Although AUX can influence ethylene (ETH) synthesis in apple (Malus domestica) fruits by affecting ETH system II, this mechanism remains to be explored. Here, we identified an ETH response factor (ERF) family transcription factor, MdERF003, whose expression could be activated by naphthalene acetic acid. The transient silencing of MdERF003 inhibited ETH synthesis in fruits, and MdERF003 could bind to the MdACS1 promoter. To explore the upstream target genes of MdERF003, we screened the MdARF family members by yeast one-hybrid assays of the MdERF003 promoter, and found that the transcription factor MdARF17, which showed AUX-promoted expression, could bind to the MdERF003 promoter and promote its expression. Finally, we silenced MdERF003 in apple fruits overexpressing MdARF17 and found that MdERF003 plays a role in MdARF17-promoted ETH synthesis in apple. Thus, AUX-MdARF17-MdERF003 promotes ETH synthesis in apple fruits.

Hypoxia represses pattern-triggered immune responses in Arabidopsis.

Biotic and abiotic stresses frequently co-occur in nature, yet relatively little is known about how plants co-ordinate the response to combined stresses. Protein degradation by the ubiquitin/proteasome system is central to the regulation of multiple independent stress response pathways in plants. The Arg/N-degron pathway is a subset of the ubiquitin/proteasome system that targets proteins based on their N termini and has been specifically implicated in the responses to biotic and abiotic stresses, including hypoxia, via accumulation of group VII ETHYLENE RESPONSE FACTOR (ERF-VII) transcription factors that orchestrate the onset of the hypoxia response program. Here, we investigated the role of the Arabidopsis (Arabidopsis thaliana) Arg/N-degron pathway in mediating the crosstalk between combined abiotic and biotic stresses using hypoxia treatments and the flg22 elicitor of pattern-triggered immunity (PTI), respectively. We uncovered a link between the plant transcriptional responses to hypoxia and flg22. Combined hypoxia and flg22 treatments showed that hypoxia represses the flg22 transcriptional program, as well as the expression of pattern recognition receptors, mitogen-activated protein kinase (MAPK) signalling and callose deposition during PTI through mechanisms that are mostly independent from the ERF-VIIs. These findings improve our understanding of the trade-offs between plant responses to combined abiotic and biotic stresses in the context of our efforts to increase crop resilience to global climate change. Our results also show that the well-known repressive effect of hypoxia on innate immunity in animals also applies to plants.

Genome-Wide Characterization and Expression Profiling of the AP2/ERF Gene Family in Fragaria vesca L.

The wild strawberry (Fragaria vesca L.; F. vesca) represents a resilient and extensively studied model organism. While the AP2/ERF gene family plays a pivotal role in plant development, its exploration within F. vesca remains limited. In this study, we characterized the AP2/ERF gene family in wild strawberries using the recently released genomic data (F. vesca V6.0). We conducted an analysis of the gene family expansion pattern, we examined gene expression in stem segments and leaves under cold conditions, and we explored its functional attributes. Our investigation revealed that the FvAP2/ERF family comprises 86 genes distributed among four subfamilies: AP2 (17), RAV (6), ERF (62), and Soloist (1). Tandem and segmental duplications significantly contributed to the growth of this gene family. Furthermore, predictive analysis identified several cis-acting elements in the promoter region associated with meristematic tissue expression, hormone regulation, and resistance modulation. Transcriptomic analysis under cold stress unveiled diverse responses among multiple FvAP2/ERFs in stem segments and leaves. Real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) results confirmed elevated expression levels of select genes following the cold treatment. Additionally, overexpression of FvERF23 in Arabidopsis enhanced cold tolerance, resulting in significantly increased fresh weight and root length compared to the wild-type control. These findings lay the foundation for further exploration into the functional roles of FvAP2/ERF genes.

The methyl jasmonate-responsive transcription factor SmERF106 promotes tanshinone accumulation in Salvia miltiorrhiza.

Understanding the regulatory biosynthesis mechanisms of active compounds in herbs is vital for the preservation and sustainable use of natural medicine resources. Diterpenoids, which play a key role in plant growth and resistance, also serve as practical products for humans. Tanshinone, a class of abietane-type diterpenes unique to the Salvia genus, such as Salvia miltiorrhiza, is an excellent model for studying diterpenoids. In this study, we discovered that a transcription factor, SmERF106, responds to MeJA induction and is located in the nucleus. It exhibits a positive correlation with the expression of SmKSL1 and SmIDI1, which are associated with tanshinone biosynthesis. We performed DNA affinity purification sequencing (DAP-seq) to predict genes that may be transcriptionally regulated by SmERF106. Our cis-elements analysis suggested that SmERF106 might bind to GCC-boxes in the promoters of SmKSL1 and SmIDI1. This indicates that SmKSL1 and SmIDI1 could be potential target genes regulated by SmERF106 in the tanshinone biosynthesis pathway. Their interaction was then demonstrated through a series of in vitro and in vivo binding experiments, including Y1H, EMSA, and Dual-LUC. Overexpression of SmERF106 in the hairy root of S. miltiorrhiza led to a significant increase in tanshinone content and the transcriptional levels of SmKSL1 and SmIDI1. In summary, we found that SmERF106 can activate the transcription of SmKSL1 and SmIDI1 in response to MeJA induction, thereby promoting tanshinone biosynthesis. This discovery provides new insights into the regulatory mechanisms of tanshinones in response to JA and offers a potential gene tool for tanshinone metabolic engineering strategy.

Functional Activity of Isoform 2 of Human eRF1.

Eukaryotic release factor eRF1, encoded by the ETF1 gene, recognizes stop codons and induces peptide release during translation termination. ETF1 produces several different transcripts as a result of alternative splicing, from which two eRF1 isoforms can be formed. Isoform 1 codes well-studied canonical eRF1, and isoform 2 is 33 amino acid residues shorter than isoform 1 and completely unstudied. Using a reconstituted mammalian in vitro translation system, we showed that the isoform 2 of human eRF1 is also involved in translation. We showed that eRF1iso2 can interact with the ribosomal subunits and pre-termination complex. However, its codon recognition and peptide release activities have decreased. Additionally, eRF1 isoform 2 exhibits unipotency to UGA. We found that eRF1 isoform 2 interacts with eRF3a but stimulated its GTPase activity significantly worse than the main isoform eRF1. Additionally, we studied the eRF1 isoform 2 effect on stop codon readthrough and translation in a cell-free translation system. We observed that eRF1 isoform 2 suppressed stop codon readthrough of the uORFs and decreased the efficiency of translation of long coding sequences. Based on these data, we assumed that human eRF1 isoform 2 can be involved in the regulation of translation termination. Moreover, our data support previously stated hypotheses that the GTS loop is important for the multipotency of eRF1 to all stop codons. Whereas helix α1 of the N-domain eRF1 is proposed to be involved in conformational rearrangements of eRF1 in the A-site of the ribosome that occur after GTP hydrolysis by eRF3, which ensure hydrolysis of peptidyl-tRNA at the P site of the ribosome.

JA/ethylene and NaWRKY6/3 regulated Alternaria resistance depends on ethylene response factor 1B-like in wild tobacco.

Biosynthesis of the phytoalexins scopoletin and scopolin in Nicotiana species is regulated by upstream signals including jasmonate (JA), ethylene (ET) and NaWRKY3 in response to the necrotrophic fungus Alternaria alternata, which causes brown spot disease. However, how these signals are coordinated to regulate these phytoalexins remains unknown. By analyzing RNA sequencing data and RNA interference, we identified NaERF1B-like (NaERF1B-L) as a key player in Nicotiana attenuata during A. alternata infection by regulating the transcripts of Feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), encoding a key enzyme for scopoletin biosynthesis, and NaVS1-like (NaVS1-L), a putative biosynthetic gene of the phytoalexin solavetivone. We further demonstrated that the synergistic induction of these two genes by JA and ET signaling is mediated by NaERF1B-L. Additionally, we found that the two closely related proteins NaWRKY6 and NaWRKY3 physically interact to enhance NaERF1B-L expression by directly binding and activating the NaERF1B-L promoter. Collectively, our current results demonstrate that NaERF1B-L plays a positive role in resistance to A. alternata by modulating phytoalexins biosynthesis through the integration of JA/ET and NaWRKY6/3 signaling. Our findings reveal a fine-tuned transcriptional regulatory hierarchy mediated by NaERF1B-L for brown spot disease resistance in wild tobacco.