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 Characterization of AP2/ERF Transcription Factors during Flower Development and Anthocyanin Biosynthesis Related Candidate Genes in Lycoris.

The APETALA2/ethylene-responsive transcription factor (AP2/ERF) family has been extensively investigated because of its significant involvement in plant development, growth, fruit ripening, metabolism, and plant stress responses. To date, there has been little investigation into how the AP2/ERF genes influence flower formation and anthocyanin biosynthesis in Lycoris. Herein, 80 putative LrAP2/ERF transcription factors (TFs) with complete open reading frames (ORFs) were retrieved from the Lycoris transcriptome sequence data, which could be divided into five subfamilies dependent on their complete protein sequences. Furthermore, our findings demonstrated that genes belonging to the same subfamily had structural similarities and conserved motifs. LrAP2/ERF genes were analyzed for playing an important role in plant growth, water deprivation, and flower formation by means of gene ontology (GO) enrichment analysis. The expression pattern of the LrAP2/ERF genes differed across tissues and might be important for Lycoris growth and flower development. In response to methyl jasmonate (MeJA) exposure and drought stress, the expression of each LrAP2/ERF gene varied across tissues and time. Moreover, a total of 20 anthocyanin components were characterized using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) analysis, and pelargonidin-3-O-glucoside-5-O-arabinoside was identified as the major anthocyanin aglycone responsible for the coloration of the red petals in Lycoris. In addition, we mapped the relationships between genes and metabolites and found that LrAP2/ERF16 is strongly linked to pelargonidin accumulation in Lycoris petals. These findings provide the basic conceptual groundwork for future research into the molecular underpinnings and regulation mechanisms of AP2/ERF TFs in anthocyanin accumulation and Lycoris floral development.

Identification of the AP2/ERF transcription factor family of Eleutherococcus senticosus and its expression correlation with drought stress.

In this study, through analysis of the genome of Eleutherococcus senticosus (ES). 228 AP2/ERF genes were identified and classified into 5 groups AP2 (47 genes), ERF (108 genes), RAV (6 genes), DREB (64 genes), and soloist (3 genes). According to the AP2/ERF classification of Arabidopsis thaliana, the ES AP2/ERF proteins were subdivided into 15 groups. The gene structure and motifs of each group of AP2/ERF in ES were highly similar, which confirmed the conservation of AP2/ERF genes. The ES AP2/ERF genes were unevenly distributed on chromosomes, and a total of four pairs of tandem repeats, and 84 co-linear gene pairs were found, so the AP2/ERF genes expanded in a fragment replication manner, and dominated by pure selection during evolution. By analyzing the transcriptome data of ES under different drought stress conditions, 87 AP2/ERF genes with differential expression were obtained, of which 10 genes with highly significant differences were further analyzed and screened for qRT-PCR validation. To the best of our knowledge, this is the first report on the AP2/ERF gene of Eleutherococcus senticosus, and the bioinformatics analysis and experimental validation provided valuable information about them, which is of great significance for further research on the molecular mechanisms of ES in response to drought stress.

Comparative transcriptomic profiling of peach and nectarine cultivars reveals cultivar-specific responses to chilled postharvest storage.

Introduction: Peach (Prunus persica (L.) Batsch,) and nectarine fruits (Prunus persica (L.) Batsch, var nectarine), are characterized by a rapid deterioration at room temperature. Therefore, cold storage is widely used to delay fruit post-harvest ripening and extend fruit commercial life. Physiological disorders, collectively known as chilling injury, can develop typically after 3 weeks of low-temperature storage and affect fruit quality. Methods: A comparative transcriptomic analysis was performed to identify regulatory pathways that develop before chilling injury symptoms are detectable using next generation sequencing on the fruits of two contrasting cultivars, one peach (Sagittaria) and one nectarine, (Big Top), over 14 days of postharvest cold storage. Results: There was a progressive increase in the number of differentially expressed genes between time points (DEGs) in both cultivars. More (1264) time point DEGs were identified in 'Big Top' compared to 'Sagittaria' (746 DEGs). Both cultivars showed a downregulation of pathways related to photosynthesis, and an upregulation of pathways related to amino sugars, nucleotide sugar metabolism and plant hormone signal transduction with ethylene pathways being most affected. Expression patterns of ethylene related genes (including biosynthesis, signaling and ERF transcription factors) correlated with genes involved in cell wall modification, membrane composition, pathogen and stress response, which are all involved later during storage in development of chilling injury. Discussion: Overall, the results show that common pathways are activated in the fruit of 'Big Top' nectarine and 'Sagittaria' peach in response to cold storage but include also differences that are cultivar-specific responses.

Genome-wide characterization and expression analysis of Erf gene family in cotton.

BACKGROUND: AP2/ERF transcription factors are important in a variety of biological activities, including plant growth, development, and responses to biotic and abiotic stressors. However, little study has been done on cotton's AP2/ERF genes, although cotton is an essential fibre crop. We were able to examine the tissue and expression patterns of AP2/ERF genes in cotton on a genome-wide basis because of the recently published whole genome sequence of cotton. Genome-wide analysis of ERF gene family within two diploid species (G. arboreum & G. raimondii) and two tetraploid species (G. barbadense, G. hirsutum) was performed. RESULTS: A total of 118, 120, 213, 220 genes containing the sequence of single AP2 domain were identified in G. arboreum, G. raimondii, G. barbadense and G. hirsutum respectively. The identified genes were unevenly distributed across 13/26 chromosomes of A and D genomes of cotton. Synteny and collinearity analysis revealed that segmental duplications may have played crucial roles in the expansion of the cotton ERF gene family, as well as tandem duplications played a minor role. Cis-acting elements of the promoter sites of Ghi-ERFs genes predict the involvement in multiple hormone responses and abiotic stresses. Transcriptome and qRT-PCR analysis revealed that Ghi-ERF-2D.6, Ghi-ERF-12D.13, Ghi-ERF-6D.1, Ghi-ERF-7A.6 and Ghi-ERF-11D.5 are candidate genes against salinity tolerance in upland cotton. CONCLUSION: Overwhelmingly, the present study paves the way to better understand the evolution of cotton ERF genes and lays a foundation for future investigation of ERF genes in improving salinity stress tolerance in cotton.

L2, a chloroplast metalloproteinase, regulates fruit ripening by participating in ethylene autocatalysis under the control of ethylene response factors.

Although autocatalytic ethylene biosynthesis plays an important role in the ripening of climacteric fruits, our knowledge of the network that promotes it remains limited. We identified white fruit (wf), a tomato mutant that produces immature fruit that are white and that ripen slowly. We found that an inversion on chromosome 10 disrupts the LUTESCENT2 (L2) gene, and that white fruit is allelic to lutescent2. Using CRISPR/Cas9 technology we knocked out L2 in wild type tomato and found that the l2-cr mutants produced phenotypes that were very similar to white fruit (lutescent2). In the l2-cr fruit, chloroplast development was impaired and the accumulation of carotenoids and lycopene occurred more slowly than in wild type. During fruit ripening in l2-cr mutants, the peak of ethylene release was delayed, less ethylene was produced, and the expression of ACO genes was significantly suppressed. We also found that exogenous ethylene induces the expression of L2 and that ERF.B3, an ethylene response factor, binds to the promoter of the L2 gene and activates its transcription. Thus, the expression of L2 is regulated by exogenous ethylene. Taken together, our results indicate that ethylene may affect the expression of L2 gene and that L2 participates in autocatalytic ethylene biosynthesis during tomato fruit ripening.

Mechanism by which salt stress induces physiological responses and regulates tanshinone synthesis.

Salvia miltiorrhiza is a traditional Chinese herbal medicine with tanshinone as one of the main bioactive components and has antitumor, antibacterial, anti-inflammatory properties, as well as other physiological functions. Tanshinone, as a secondary metabolite, is synthesized under salt stress or other environmental stresses. Oxidative stress is an important physiological response of plants to salt stress. Transcription factors (TFs) are believed to play regulatory roles in this process, and AP2/ERF TFs have significant effects on defense against the adversity of plants. However, investigations on the regulation of AP2/ERF TFs in tanshinone synthesis under salt stress are limited. In this research, the tanshinone content, related gene expression and activities of enzymes, and the markers of oxidative stress were determined. The results showed that SmAP1, SmAP2 and SmERF2 were AP2/ERF TFs with AP conserved sequences, whose relative expression levels increased and were positively correlated with the contents of tanshinone I (T-I), tanshinone IIA (T-IIA) and cryptotanshinone (CT) in the roots of Salvia miltiorrhiza. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) increased in the roots of Salvia miltiorrhiza. The expression levels of genes encoding enzymes and the activities of key enzymes in the tanshinone biosynthesis pathway increased accordingly. The results showed that AP2/ERF TFs could positively regulate the biosynthesis of tanshinone in the roots of Salvia miltiorrhiza under salt stress.

An amiRNA screen uncovers redundant CBF and ERF34/35 transcription factors that differentially regulate arsenite and cadmium responses.

Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional regulators of arsenic-induced gene expression in plants remain less well known. To date, forward genetic screens have proven limited for dissecting arsenic response mechanisms. We hypothesized that this may be due to the extensive genetic redundancy present in plant genomes. To overcome this limitation, we pursued a forward genetic screen for arsenite tolerance using a randomized library of plants expressing >2,000 artificial microRNAs (amiRNAs). This library was designed to knock-down diverse combinations of homologous gene family members within sub-clades of transcription factor and transporter gene families. We identified six transformant lines showing an altered response to arsenite in root growth assays. Further characterization of an amiRNA line targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation of CRISPR lines, higher-order T-DNA mutants and gene expression analyses, further support our findings. These ERF transcription factors differentially regulate arsenite sensitivity and cadmium tolerance.

[Research progress on effect of AP2/ERF transcription factors in regulating secondary metabolite biosynthesis].

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.

Genome-wide identification and expression analysis of the ERF transcription factor family in pineapple (Ananas comosus (L.) Merr.).

Pineapple (Ananas comosus (L.) Merr.) is an important tropical fruit with high economic value. The quality and yield of pineapple will be affected by various environmental conditions. Under adverse conditions, plants can produce a complex reaction mechanism to enhance their resistance. It has been reported that the member of ethylene responsive transcription factors (ERFs) plays a crucial role in plant developmental process and stress response. However, the function of these proteins in pineapple remains limited. In this study, a total of 74 ERF genes (AcoERFs) were identified in pineapple genome, named from AcoERF1 to AcoERF74, and divided into 13 groups based on phylogenetic analysis. We also analyzed gene structure, conserved motif and chromosomal location of AcoERFs, and the AcoERFs within the same group possess similar gene structures and motif compositions. Three genes (AcoERF71, AcoERF73 and AcoERF74) were present on unanchored scaffolds, so they could not be conclusively mapped on chromosome. Synteny and cis-elements analysis of ERF genes provided deep insight into the evolution and function of pineapple ERF genes. Furthermore, we analyzed the expression profiling of AcoERF in different tissues and developmental stages, and 22 AcoERF genes were expressed in all examined tissues, in which five genes (AcoERF13, AcoERF16, AcoERF31, AcoERF42, and AcoERF65) had high expression levels. Additionally, nine AcoERF genes were selected for functional verification by qRT-PCR. These results provide useful information for further investigating the evolution and functions of ERF family in pineapple.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

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.

ETHYLENE RESPONSE FACTOR 115 integrates jasmonate and cytokinin signaling machineries to repress adventitious rooting in Arabidopsis.

Adventitious root initiation (ARI) is a de novo organogenesis program and a key adaptive trait in plants. Several hormones regulate ARI but the underlying genetic architecture that integrates the hormonal crosstalk governing this process remains largely elusive. In this study, we use genetics, genome editing, transcriptomics, hormone profiling and cell biological approaches to demonstrate a crucial role played by the APETALA2/ETHYLENE RESPONSE FACTOR 115 transcription factor. We demonstrate that ERF115 functions as a repressor of ARI by activating the cytokinin (CK) signaling machinery. We also demonstrate that ERF115 is transcriptionally activated by jasmonate (JA), an oxylipin-derived phytohormone, which represses ARI in NINJA-dependent and independent manners. Our data indicate that NINJA-dependent JA signaling in pericycle cells blocks early events of ARI. Altogether, our results reveal a previously unreported molecular network involving cooperative crosstalk between JA and CK machineries that represses ARI.

Comparative transcriptome analysis of MeJA-responsive AP2/ERF transcription factors involved in notoginsenosides biosynthesis.

Differential transcriptome analysis is an effective method for gene selection of triterpene saponin biosynthetic pathways. MeJA-induced differential transcriptome of Panax notoginseng has not been analyzed yet. In this study, comparative transcriptome analysis of P. notoginseng roots and methyl jasmonate (MeJA)-induced roots revealed 83,532 assembled unigenes and 21,947 differentially expressed unigenes. Sixteen AP2/ERF transcription factors, which were significantly induced by MeJA treatment in the root of P. notoginseng, were selected for further analysis. Real-time quantitative PCR (RT-qPCR) and co-expression network analysis of the 16 AP2/ERF transcription factors showed that PnERF2 and PnERF3 had significant correlation with dammarenediol II synthase gene (DS) and squalene epoxidase gene (SE), which are key genes in notoginsenoside biosynthesis, in different tissues and MeJA-induced roots. A phylogenetic tree was conducted to analyze the 16 candidate AP2/ERF transcription factors and other 38 transcription factors. The phylogenetic tree analysis showed PnERF2, AtERF3, AtERF7, TcERF12 and other seven transcriptional factors are in same branch, while PnERF3 had close evolutionary relationships with AtDREB1A, GhERF38 and TcAP2. The results of comparative transcriptomes and AP2/ERF transcriptional factors analysis laid a solid foundation for further investigations of disease resistance and notoginsenoside biosynthesis in P. notoginseng.

AP2/ERF transcription factors regulate salt-induced chloroplast division in the moss Physcomitrella patens.

Chloroplast division is a critical process for the maintenance of appropriate chloroplast number in plant cells. It is known that in some plant species and cell types, environmental stresses can affect chloroplast division, differentiation and morphology, however the significance and regulation of these processes are largely unknown. Here we investigated the regulation of salt stress-induced chloroplast division in protonemal cells of the moss, Physcomitrella patens, and found that, salt stress as one of the major abiotic stresses, induced chloroplast division and resulted in increased chloroplast numbers. We further identified three APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors (TFs) that were responsible for this regulation. These AP2/ERF genes were up-regulated under salt stress, and amino acid sequences and phylogenetic analyses indicated that all TFs possess only one conserved AP2 domain and likely belong to the same subgroup of ERF-B3 in the AP2/ERF superfamily. Overexpression of these TFs significantly increased the chloroplast number even in the absence of NaCl stress. On the contrary, inducible overexpression of the dominant repressor form of these TFs suppressed salt stress-induced chloroplast division. Thus, our results suggest that salt stress induced-chloroplast division is regulated through members of the AP2/ERF TF superfamily.

Overexpression of Barley Transcription Factor HvERF2.11 in Arabidopsis Enhances Plant Waterlogging Tolerance.

Waterlogging stress significantly affects the growth, development, and productivity of crop plants. However, manipulation of gene expression to enhance waterlogging tolerance is very limited. In this study, we identified an ethylene-responsive factor from barley, which was strongly induced by waterlogging stress. This transcription factor named HvERF2.11 was 1158 bp in length and encoded 385 amino acids, and mainly expressed in the adventitious root and seminal root. Overexpression of HvERF2.11 in Arabidopsis led to enhanced tolerance to waterlogging stress. Further analysis of the transgenic plants showed that the expression of AtSOD1, AtPOD1 and AtACO1 increased rapidly, while the same genes did not do so in non-transgenic plants, under waterlogging stress. Activities of antioxidant enzymes and alcohol dehydrogenase (ADH) were also significantly higher in the transgenic plants than in the non-transgenic plants under waterlogging stress. Therefore, these results indicate that HvERF2.11 plays a positive regulatory role in plant waterlogging tolerance through regulation of waterlogging-related genes, improving antioxidant and ADH enzymes activities.

The Role of Major Transcription Factors in Solanaceous Food Crops under Different Stress Conditions: Current and Future Perspectives.

Plant growth, development, and productivity are adversely affected by environmental stresses such as drought (osmotic stress), soil salinity, cold, oxidative stress, irradiation, and diverse diseases. These impacts are of increasing concern in light of climate change. Noticeably, plants have developed their adaptive mechanism to respond to environmental stresses by transcriptional activation of stress-responsive genes. Among the known transcription factors, DoF, WRKY, MYB, NAC, bZIP, ERF, ARF and HSF are those widely associated with abiotic and biotic stress response in plants. Genome-wide identification and characterization analyses of these transcription factors have been almost completed in major solanaceous food crops, emphasizing these transcription factor families which have much potential for the improvement of yield, stress tolerance, reducing marginal land and increase the water use efficiency of solanaceous crops in arid and semi-arid areas where plant demand more water. Most importantly, transcription factors are proteins that play a key role in improving crop yield under water-deficient areas and a place where the severity of pathogen is very high to withstand the ongoing climate change. Therefore, this review highlights the role of major transcription factors in solanaceous crops, current and future perspectives in improving the crop traits towards abiotic and biotic stress tolerance and beyond. We have tried to accentuate the importance of using genome editing molecular technologies like CRISPR/Cas9, Virus-induced gene silencing and some other methods to improve the plant potential in giving yield under unfavorable environmental conditions.

Isolation, sequencing, and expression analysis of 30 AP2/ERF transcription factors in apple.

BACKGROUND: AP2/ERF transcription factors are involved in the regulation of plant growth, development, and stress responses. Our research objective was to characterize novel apple (Malus × domestica Borkh.) genes encoding AP2/ERF transcription factors involved in regulation of plant growth, development, and stress response. The transcriptional level of apple AP2/ERF genes in different tissues and under various biotic and abiotic stress was determined to provide valuable insights into the function of AP2/ERF transcription factors in apple. METHODS: Thirty full-length cDNA sequences of apple AP2/ERF genes were isolated from 'Zihong Fuji' apple (Malus × domestica cv. Zihong Fuji) via homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. Expression levels of apple AP2/ERF genes were detected in 16 different tissues using a known array. Expression patterns of apple AP2/ERF genes were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq with existing data, and the expression of apple AP2/ERF genes was analyzed under NaCl and mannitol treatments using qRT-PCR. RESULTS: The sequencing results produced 30 cDNAs (designated as MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF31-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2). Phylogenetic analysis revealed that MdERF11/16, MdERF33/35, MdERF34/39, and MdERF18/23 belonged to groups A-2, A-4, A-5, and A-6 of the DREB subfamily, respectively; MdERF31, MdERF19, MdERF4/25/28/32, MdERF24, MdERF5/6/27, and MdERF3/7/8/17/22/26 belonged to groups B-1, B-2, B-3, B-4, B-5, and B-6 of the ERF subfamily, respectively; MdAP2D60 and MdAP2D62/63/64/65 belonged to the AP2 subfamily; and MdRAV2 belonged to the RAV subfamily. Array results indicated that 30 apple AP2/ERF genes were expressed in all examined tissues to different degrees. RNA-seq results using previously reported data showed that many members of the apple ERF and DREB subfamilies were induced by Alternaria alternate apple pathotype (AAAP) infection. Under salt treatment, many members in the apple ERF and DREB subfamilies were transcriptionally up or down-regulated. Under mannitol treatment, many members of the apple ERF, DREB, and AP2 subfamilies were induced at the transcriptional level. Taken together, the results indicated that the cloned apple AP2/ERF genes were expressed in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol treatment, which suggested they may be involved in regulating growth, development, and stress response in apple.

Computational Disorder Analysis in Ethylene Response Factors Uncovers Binding Motifs Critical to Their Diverse Functions.

APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERFs) play crucial roles in adaptation to stresses such as those caused by pathogens, wounding and cold. Although their name suggests a specific role in ethylene signalling, some ERF members also co-ordinate signals regulated by other key plant stress hormones such as jasmonate, abscisic acid and salicylate. We analysed a set of ERF proteins from three divergent plant species for intrinsically disorder regions containing conserved segments involved in protein-protein interaction known as Molecular Recognition Features (MoRFs). Then we correlated the MoRFs identified with a number of known functional features where these could be identified. Our analyses suggest that MoRFs, with plasticity in their disordered surroundings, are highly functional and may have been shuffled between related protein families driven by selection. A particularly important role may be played by the alpha helical component of the structured DNA binding domain to permit specificity. We also present examples of computationally identified MoRFs that have no known function and provide a valuable conceptual framework to link both disordered and ordered structural features within this family to diverse function.

The ethylene response factor SmERF6 co-regulates the transcription of SmCPS1 and SmKSL1 and is involved in tanshinone biosynthesis in Salvia miltiorrhiza hairy roots.

MAIN CONCLUSION: The SmERF6, which recognizes the GCC-box of SmCPS1 and SmKSL1 promoter in nucleus, regulates the tanshinone biosynthesis in Salvia miltiorrhiza hairy roots. Tanshinone, an important medicinal ingredient in Salvia miltiorrhiza, is best known for its use in medicine. However, the transcription factor regulation of tanshinone biosynthesis is unclear. Here, we isolated and identified a transcription factor in the ERF family of S. miltiorrhiza, SmERF6, which was screened from an S. miltiorrhiza cDNA library by the promoters of two key tanshinone synthesis genes (SmKSL1 and SmCPS1); this factor regulated tanshinone biosynthesis. The gene was highly expressed in the root and responded to ethylene treatment. SmERF6 modulated tanshinone biosynthesis by directly binding to an ethylene-responsive element (GCC-box) of the SmKSL1 and SmCPS1 promoters and activating their transcription. Overexpression of SmERF6 in the hairy roots increased their tanshinone accumulation, and SmERF6 silencing by RNAi led to a lower tanshinone content. Furthermore, tanshinone accumulation maintained homeostasis with the total phenolic acid and flavonoid contents in S. miltiorrhiza. These findings elucidated how SmERF6 directly co-regulates the transcription of SmCPS1 and SmKSL1 and modulates tanshinone synthesis to accelerate the metabolic flux of tanshinone accumulation in S. miltiorrhiza.