The ERF transcription factor LTF1 activates DIR1 to control stereoselective synthesis of antiviral lignans and stress defense in Isatis indigotica roots

Lignans are a powerful weapon for plants to resist stresses and have diverse bioactive functions to protect human health. Elucidating the mechanisms of stereoselective biosynthesis and response to stresses of lignans is important for the guidance of plant improvement. Here, we identified the complete pathway to stereoselectively synthesize antiviral (-)-lariciresinol glucosides in Isatis indigotica roots, which consists of three-step sequential stereoselective enzymes DIR1/2, PLR, and UGT71B2. DIR1 was further identified as the key gene in respoJanuary 2024nse to stresses and was able to trigger stress defenses by mediating the elevation in lignan content. Mechanistically, the phytohormone-responsive ERF transcription factor LTF1 colocalized with DIR1 in the cell periphery of the vascular regions in mature roots and helped resist biotic and abiotic stresses by directly regulating the expression of DIR1. These systematic results suggest that DIR1 as the first common step of the lignan pathway cooperates with PLR and UGT71B2 to stereoselectively synthesize (-)-lariciresinol derived antiviral lignans in I. indigotica roots and is also a part of the LTF1-mediated regulatory network to resist stresses. In conclusion, the LTF1-DIR1 module is an ideal engineering target to improve plant Defenses while increasing the content of valuable lignans in plants.

Identification and Expression Analysis of AP2/ERF Family in Stress Responses of Pinellia ternata Based on Transcriptome Data / 中国实验方剂学杂志

ObjectiveTo identify the functions of the AP2/ERF family members in Pinellia ternata and promote the genetic improvement of P. ternata varieties. MethodWe identified and conducted a systematic bioinformatics analysis of the AP2/ERF family member genes in P. ternata based on the three generations of transcriptome data. Real-time polymerase Chain reaction　（Real-time） PCR was employed to determine the expression pattern of AP2/ERF genes in different tissues and under different stress conditions. ResultA total of eight full-length AP2/ERF family members were identified from the transcriptome data， which were classified into three sub-gene families： AP2， ERF， and DREB. The deduced AP2/ERF proteins in P. ternata had the length of 251-512 aa， the theoretical pI of 5.29-11.72， the instability index of 45.90-82.41， subcellular localization in the nucleus， and conserved domains and motifs. AP2/ERF genes were expressed in different tissues of P. ternata， with high expression levels in the leaf. The stress response experiments showed that PtERF1 mainly responded to NaCl stress. The expression of PtERF2 and PtERF4 was significantly up-regulated under low temperature and polyethylene glycol （PEG）-simulated stress. PtERF3 responded to both low temperature and NaCl stress. The expression of PtERF5 was induced by high temperature， low temperature， NaCl and PEG stress. The expression of PtERF7 was up-regulated under high temperature， while that of PtERF8 under low temperature. ConclusionThe AP2/ERF genes in P. ternata can respond to stress and have the potential functions of regulating photosynthesis and improving root stress resistance.

Identification and analysis of AP2/ERF gene family of Panax notoginseng and function of PnDREB84 gene / 药学学报

The AP2/ERF gene family is one of the largest transcription factor families in the plant kingdom, and plays an important role in response to biological and abiotic stresses, plant hormone responses, and plant growth and development. In this study, the AP2/ERF family of Panax notoginseng was identified by bioinformatics methods, and the physicochemical properties, structure, phylogenetic relationship, expression pattern and function of PnDREB4 gene of the family were analyzed. The results showed that 140 AP2/ERF family members were identified in P. notoginseng, which were divided into DREB, ERF, AP2, RAV and Sololit subgroups. The physicochemical properties and motifs of proteins were similar among the subgroups. There were 34 differentially expressed genes in the AP2/ERF family of Fusarium oxysporum infected P. notoginseng plants, and 19 genes were up-regulated. The expression level of PnDREB84 was up-regulated with the extension of Fusarium oxysporum infection time in the range of 0-96 h. The content of ABA and SA in P. notoginseng plants overexpressing PnDREB84 gene increased after 4 ℃ stress. The results showed that PnDREB84 gene plays a dual regulatory role in the process of biological stress and abiotic stress. PnDREB84 gene can be used as a potential molecular marker for the breeding of new varieties of P. notoginseng. The identification of AP2/ERF transcription factor and function analysis of PnDREB84 gene of P. notoginseng provided data support for the analysis of stress resistance mechanism of P. notoginseng and the breeding of new varieties.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.@*METHODS@#To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.@*RESULTS@#One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.@*CONCLUSION@#This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata / 中国天然药物

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Expression analysis of transcription factor ERF gene family of Panax ginseng / 中国中药杂志

Ethylene responsive factor(ERF), one of the largest families of transcriptional factors in plants, plays a key role in se-condary metabolism of herbal plants. To analyze the expression of ERF family genes, the heat map clustering method was used by analyzing the ginseng transcriptomes of different parts and different growth years. The contents of ginsenosides Rg_1, Re and Rb_1 in various concentrations of MeJA-treated ginseng adventitious roots were determined by UPLC-MS/MS method. The expression of key genes of ginsenoside biosynthesis(DDS, CYP716A47, CYP716A53v2) and ERF family genes in MeJA-treated ginseng adventitious roots were determined by using real-time quantitative PCR. Pearson correlation was adopted to analyze the gene expression pattern of DDS, CYP716A47, CYP716A53v2 gene and ERF family. The results showed that the content of ginseng diol ginsenoside Rb_1 in ginseng adventitious roots treated with different concentrations of MeJA increased, and the content of ginseng triol ginsenoside Rg_1 and Re decreased. It is consistent with the increase of DDS and CYP716A47 expression and the decrease of CYP716A53v2 gene expression. The expression of ERF003, ERF118 and ERF012 genes was significantly positively correlated with CYP716A53v2, but negatively correlated with DDS. While the expression of ERF1B was significantly negatively correlated with CYP716A47.It is proved that ERF003, ERF118 and ERF012 were likely to inhibit the expression of DDS and promote the expression of CYP716A53v2, and ERF1B was likely to inhibit CYP716A47. This work could provide theoretical basis of ERF functional verification of regulating the biosynthesis of ginsenosides.

Research progress on effect of AP2/ERF transcription factors in regulating secondary metabolite biosynthesis / 中国中药杂志

AP2/ERF transcription factor is a kind of transcription factors widely existing in plants, and contains at least a conserved AP2/ERF domain composed of about 60-70 amino acids. AP2/ERF transcription factors are widely involved in a variety of physiological processes in plants, including plant development, fruit ripening, flower development and other plant development processes, as well as such stress response processes as damage, pathogen defense, high-salt condition and drought. In recent years, secondary metabolic engineering that takes transcription factors as genetic manipulation targets has developed rapidly in improving the content of active ingredients and the quality of medicinal plants. This paper reviews the recent progress in the regulation of secondary metabolites biosynthesis with AP2/ERF transcription factors, and provides theoretical basis for the exploration of efficient regulatory targets, the regulation of secondary metabolites in medicinal plants, the targeted improvement of the content of active ingredients in traditional Chinese medicine, and the sustainable supply of high-quality traditional Chinese medicines.

Cloning of ERF1 transcription factor from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods: A gene (CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35S promoter. Results: CtERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The pBASTA-CtERF1 plant expression vector was constructed successfully by molecular biology. Conclusion: A CtERF1 gene of C. tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector pBASTA-CtERF1 was constructed successfully.

In Silico Cloning and Bioinformatics Analysis of AP2/ERF Family Gene from Brassica Oleracea / 中国药师

Objective:To obtain AP2/ERF genes from Brassica oleracea by using in silico cloning. Methods: AP2/ERF genes were cloned by retnieving the EST database and using the bioinformatics software with the Arabidopsis thaliana AP2/ERF as a querying probe. Results:Two AP2/ERF family transcriptional regulators (BoAP2/ERF1 and BoAP2/ERF2) were isolated from Brassica olera-cea by the in silico cloning method. Some characters of AP2/ERF protein were analyzed and predicted by the tools of bioinformatics in the following aspects including the composition of amino acid sequence, hydrophilicity and hydrophobicity, subcellular localization, secondary and tertiary structure of protein and function. Conclusion:Bioinformatical analysis shows BoAP2/ERF1 and BoAP2/ERF2 gene encode 40. 85kDa and 39. 44kDa protein with 371 and 352 amino acids. The domain is predicted to locate on nucleus. Sequence analysis indicates the protein may be involved in signaling transducer and stressing response roles in plantbiotic stresses.

Expression analysis of bZIP transcription factor encoding genes in response to water deficit stress in rice.

In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate several developmental processes and activate genes in response to biotic and abiotic stresses. Role of stress responsive bZIP transcription factors was studied in paddy in relation to different stages of development and water deficit stress (WDS) in a drought tolerant cultivar N22 and susceptible IR 64. Further, relative water content (RWC), membrane stability index (MSI) and abscisic acid (ABA) content were measured as indices of WDS at different stages of development and levels of stress. Expression of stress responsive bZIP transcription factors was directly correlated to developmental stage and WDS and indirectly to RWC, MSI and ABA content.

Cloning and characterization of a water deficit stress responsive transcription factor gene from Oryza sativa L.

Understanding the biochemical and molecular basis of drought mechanism in rice is important as drought is one of the major causes affecting rice crop adversely. A 1017 bp gene sequence encoding AP2/ERF family TF was isolated from Oryza sativa sp. Indica cv N22 encoding a protein of 338 amino acid residues, with a molecular weight of 36.58 kDa, and no intron in the ORF. The gene was named as AP2/ERF-N22(2) different from the drought responsive gene AP2/ERF-N22 that we reported earlier. AP2/ERF-N22(2) has entirely different characteristics from that of AP2/ERF-N22. It has a single AP2 domain of 55 amino acid residues and a cluster of acidic amino acid residues at the C-terminal region, which could function as a trans-activation domain. Presence of NLS indicates that it is a nuclear localized transcription factor encoding gene. It falls in group VI L, sharing characteristic similarities. Arabidopsis members of group VI L have been shown to be involved in response to cytokinin under drought stress.

Research progress on AP2/ERF transcription factors regulating biosynthesis of active ingredients in medicinal plants / 中草药

AP2/ERF transcription factors are widely present in plants. Each member of AP2/ERF family contains a much conserved DNA binding domain, AP2/EREBP binding domain, which is composed by about 60 amino acids. The studies about plant AP2/ERF transcription factors mainly focused on the developmental and physiological regulation function. In recent years, the function of regulating secondary metabolites is being concerned. Here the research progress on AP2/ERF regulating the biosynthesis of active ingredient of medicinal plants was reviewed, which could be used for reference in further studies on improving the accumulation of active ingredients in medicinal plants by applying metabolic engineering.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.

Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers

Members of the ERF transcription-factor family participate in a number of biological processes, viz., responses to hormones, adaptation to biotic and abiotic stress, metabolism regulation, beneficial symbiotic interactions, cell differentiation and developmental processes. So far, no tissue-expression profile of any cucumber ERF protein has been reported in detail. Recent completion of the cucumber full-genome sequence has come to facilitate, not only genome-wide analysis of ERF family members in cucumbers themselves, but also a comparative analysis with those in Arabidopsis and rice. In this study, 103 hypothetical ERF family genes in the cucumber genome were identified, phylogenetic analysis indicating their classification into 10 groups, designated I to X. Motif analysis further indicated that most of the conserved motifs outside the AP2/ERF domain, are selectively distributed among the specific clades in the phylogenetic tree. From chromosomal localization and genome distribution analysis, it appears that tandem-duplication may have contributed to CsERF gene expansion. Intron/exon structure analysis indicated that a few CsERFs still conserved the former intron-position patterns existent in the common ancestor of monocots and eudicots. Expression analysis revealed the widespread distribution of the cucumber ERF gene family within plant tissues, thereby implying the probability of their performing various roles therein. Furthermore, members of some groups presented mutually similar expression patterns that might be related to their phylogenetic groups.