Systematic analysis of the Capsicum ERF transcription factor family: Identification of regulatory factors involved in the regulation of species-specific metabolites

- Systematic analysis of the Capsicum ERF transcription factor family: identification of regulatory factors involved in the.
- However, there is little understanding of how ERFs participate in the regulatory networks of capsorubin and capsaicinoids biosynthesis..
- Results: In this study, a total of 142 ERFs were identified in the Capsicum annuum genome.
- The expression patterns of CaERF82, CaERF97, CaERF66, CaERF107 and CaERF101, which were found in cluster C9 and cluster C10, were consistent with those of accumulating of carotenoids ( β -carotene, zeaxanthin and capsorubin) in the pericarp.
- In cluster L3 and cluster L4, the expression patterns of CaERF102, CaERF53, CaERF111 and CaERF92 were similar to those of the accumulating capsaicinoids.
- Conclusion: This study will provide an extremely useful foundation for the study of candidate ERFs in the regulation of carotenoids and capsaicinoids biosynthesis in peppers..
- Full list of author information is available at the end of the article.
- In the final step, geranylgera- nyl pyrophosphate (GGPP), the prenyl lipid precursor, is transformed into capsorubin or capsanthin through a series of enzymatic actions mediated by phytoene syn- thase (PSY), phytoene desaturase (PDS), and lycopene β- cyclase (LCYB) [1, 2].
- Moreover, one of the most important characteristics of pepper fruit is pungency, which is a result of the accumulation of capsaicinoids, which are alkaloids.
- Capsaicinoids biosynthesis occurs in the epidermis of the placenta, and capsaicinoids are stored in vesicles on the surface of this tissue and the pericarp [7].
- Both capsaicin and dihydrocapsaicin were the most abundant capsaicinoids, representing 91% of the total capsacinoids content [10].
- The ERF family belongs to the largest branch of the AP2/ERF superfamily.
- For example, in the ERF subfamily, the genes in the promoter region specifically bind to the additional nucleotide acid se- quence AGCCCGCC of the GCC-box, while the.
- members of the DREB subfamily typically bind to a core sequence CCGAC which belongs to a component of dehydration-responsive element-binding [15, 16]..
- ERF TFs specifically participate in primary and second- ary metabolism of the plant as well, which includes the production of steroidal glycoalkaloids [23, 24], anthocya- nin [25] and carotenoids [18].
- The members of the ERF family are TFs that are candidates for the control capsai- cinoids biosynthesis.
- PAL genes possess a homologue of the GCC-box in their promoters, and ERF genes can combine with this cis-acting element [28, 29].
- Erf and Jerf therefore presumably participate in the capsaicinoids biosynthetic pathway.
- To obtain an understanding of the role of the ERF family in transcrip- tionally modulating carotenoids and capsaicinoids bio- synthesis, the all members of the ERF family were characterized by utilizing the newly sequenced Capsicum annuum genome.
- Over- all, this study contributed to the understanding of the function of ERF family members in the carotenoids and capsaicinoids biosynthetic pathways in peppers.
- Before phylogeny analysis was performed, multiple alignment analyses were performed using the amino acid sequences of the AP2 domains.
- The DREB subfamily was com- pletely conserved in V15 and E20, and more than 95% of the members of groups V to IX in the ERF subfamily contained A14 and D19 (Fig.
- However, the alignment revealed that the N- terminal regions of the AP2 domains in the ERF subfam- ily possessed a high homology, while those of the C- terminal regions showed very low conservation (Fig.
- Nevertheless, taking into account the topology of the tree in Fig.
- Phylogenetic analysis of the ERF family in four plant species.
- To clarify the phylogenetic relationships, an unrooted phylogenetic tree was constructed for all of the identified CaERF sequences based on their alignment with those in Arabidopsis by a neighbour-joining phylogenetic ana- lysis.
- According to the cited stud- ies [15] and taking into account the topology of the tree, the two subfamilies were further defined as 11 groups named group I to XI (Table 1.
- The height of the amino acid indicates the frequency observed in multiple alignment.
- The topology of the phylogeny was mostly similar to that tree obtained when using only protein sequences from pepper and Arabidopsis (Fig..
- To evaluate the biological functions of the CaERF pro- tein of the groups, the functional characteristics of ERF from Arabidopsis, tomato and pepper were investigated in the literature.
- As shown in Table S4, the members of the same group possessed similar biological functions, and group VIII members were found to be likely in- volved in alkaloid biosynthesis.
- Because of the import- ance of capsaicinoids and capsorubin in pepper, the possibility of the Capsicum annuum genome (version 2.0) containing putative ERF homologs involved in sec- ondary metabolites was investigated.
- A previous study demonstrated that Erf and Jerf in peppers were involved in the regulation of the pungency phenotype [31].
- Erf and Jerf were mapped to CaERF53 and CaERF101 in the Capsicum annuum genome (version 2.0), respectively..
- 2 Neighbour-joining phylogeny of the pepper ERF family in relation to Arabidopsis.
- however, both motif 2 and motif 5 were mainly shared within group VIII in the ERF subfamily (Fig.
- The remaining motifs (motif 6 to motif 15) were distrib- uted outside of the AP2 domain and were classified as.
- 3 Phylogenetic relationships and conserved motif distributions of the DREB (a) and ERF (b) subfamilies of proteins.
- Different color shapes indicated different groups in the phylogeny of CaERFs associated with Arabidopsis.
- processes, the expression patterns of CaERFs and the genes involved in synthesis in the pericarp and placenta (including and 48 DPA stages) were investigated (Fig.
- RNA-Seq raw data were retrieved from a public database [13] and all of the reads were re- mapped to the Capsicum annuum genome (version 2.0)..
- The expression of the relevant capsorubin synthesis gene gradually increased at 36 DPA, and capsorubin itself pri- marily accumulated at this stage (Fig.
- A total of 38 CaERFs (26%) were expressed at a level that could not be de- tected in any of the developmental stages of the pla- centa.
- Generally, CaERF in the same phylogenetic group revealed distinct expression.
- In the ten clusters, only the expression of members in cluster.
- 4 Transcript abundance of genes involved in capsanthin/capsaicinoids and biosynthesis and CaERF genes in the pericarp and placenta at different developmental stages.
- a and b indicate the expression patterns of genes involved in capsanthin biosynthesis and CaERF genes, respectively, in the pericarp at different developmental stages.
- The name of each gene with the name of the phylogenetic group is shown at the right of the heat map.
- Additionally, the members of two putative ‘pepper- specific groups’ (IXb and Xb) were barely expressed dur- ing all of the developmental pericarp and placenta stages, with the exception of CaERF67, CaERF73, CaERF127, and CaERF129, which exhibited low expres- sion in group IXb.
- the RPKM values were published instead, and when they were mapped to the Capsicum annuum genome (version 1.5), the expression of CaERFs clearly exhibited no tissue specificity (Fig.
- 5 Expression patterns of five CaERF TFs in different tissues and developmental stages of the pericarp.
- capsorubin started to increasingly accumulate in peri- carp tissue at the MG stage, whereas lutein content in- cluding the branch of the non-synthetic capsorubin was decreased.
- 6 Expression patterns of five CaERF genes in different tissues and developmental stages of the placenta.
- b Expression of 5 selected CaERFs associated with capsaicinoids biosynthesis in the root, stem, level, flower, seed, placenta (16DPA), pericarp (16DPA) and in seven developmental pericarp stages.
- The AP2/ERF superfamily is one of the largest TF fam- ilies in the plant kingdom, and it has been successfully identified and investigated in many plant species of se- quenced genomes [35 – 37].
- [38], they indicated that CaAP2/ERFs might be involved in the response to P.
- study of the Capsicum genome contributes to understanding the structure of gene families and pre- dicting their biological functions.
- Add- itionally, alignment analyses showed that the members of the ERF and DREB subfamilies possessed a specific WLG motif, as observed in the report of Cui et al.
- The classification of the tree was de- fined and annotated based on the proposed by Nakano et al.
- This re- sult was similar to that of Jin’s study in peppers [38], no matter the topology or classification of the tree.
- However, the members of these TFs were rarely expressed both in the pericarp and placenta throughout different developmental stages..
- Some reports suggested that the D(I/V) QAA sequences were regarded as the basic char- acteristics for the DREB family in cauliflower [36, 47], whereas motif 8 contained these conserved sequences, and it was primarily restricted to groups VI and X of the ERF family (Fig.
- Indeed, groups VI and X in the phylogenetic tree were near the branch of the DREB family (Fig.
- In some cases, the same phylogenetic subgroup had a similar transcript level [48], implying that members of the same phylogenetic subgroup might perform similar functions.
- Because their expression patterns exhibited good agreement with the transcriptional level of the cap- sorubin synthesis gene (Fig.
- These results indicated that the genes of the same phylogenetic subgroup exhibited dis- tinct expression patterns, which is consistent with the observation from a previous study [48, 49].
- Thus, it is likely that CaERF101 also regulates secondary metabolic pathways in the ripening pericarp, and the members of cluster C9 and cluster C10 are involved in carotenoids biosynthesis..
- It is likely that the members of the group containing CaERF53 and CaERF101 (VII) regulates capsaicinoids or secondary metabolite biosynthesis.
- a total of 142 members in the ERF family were identified in the pepper, and they were divided into DREB and ERF subfamilies.
- The phylogenetic analysis of the ERF family resulted in a distribution of 11 groups, of which the DREB subfamily included group I to group IV, and the ERF subfamily contained group V to group XI.
- Motifs 1 to 5 are present in the largest number of CaERF proteins and were thus designated.
- These five genes not only showed a trend that was similar to that of the accumula- tion of carotenoids biosynthesis genes (β-carotene, zea- xanthin and capsorubin) in pericarp tissue, but also were expressed at low levels in other tissues.
- Identification of ERFs in the pepper genome.
- ERF genes were retrieved from the latest version, 2.0 of the Capsicum annuum genome.
- Multiple sequence alignment and phylogenetic analysis All of the ERF amino acid sequences were aligned using Clustal X 2.1 (http://www.clustal.org/) with the default parameters.
- stem and leaf were collected, immediately frozen in li- quid nitrogen and stored at − 80 °C, some of the plants were cultivated in greenhouse conditions with a mixture substrate and were fertilized every week with water- soluble fertilizer (N: P: K Plant-Soul, China)..
- Flowers in the fifth node underwent artificial pollination at zero DPA.
- Additionally, CA00g52149 and CA12g20490 (ID in version 1.55 of the Capsicum annuum genome) were used as housekeeping genes.
- they were identified in the pepper genome and the data were unpublished.
- These samples were extracted for 12 h at room temperature.1 millilitre of the supernatant was collected and filtered through a 0.22 μm millipore membrane, and then the capsaicinoids content was determined by an HPLC system (Alliance E2695, Waters, America)..
- A total of 0.5 g of the freeze-dried samples were added to 50 ml cen- trifuge tubes with 8 ml of extracting solution containing hexyl hydride, acetone and absolute ethyl alcohol (2:1:1, HPLC grade), and then the samples underwent ultrasonic-assisted extraction for 30 min.
- Five millilitres of the supernatants was transferred into 50 ml centrifuge tubes and were mixed with 5 ml of extraction solution..
- One millilitre of the supernatant was used to determine carotenoids con- tent by an HPLC system (Alliance E2695, Waters, America)..
- Multiple sequence alignment of the DREB and ERF protein subfamilies.
- Phylogenetic tree of the pepper ERF family in relation to Arabidopsis.
- Phylogenetic tree of the pepper ERF family in relation to tomato (137), rice (138) and.
- CaERF genes identified and characterized in the pep- per.
- The funding organizations had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript..
- Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species.
- Genome-wide analysis of the ERF gene family in Arabidopsis and rice.
- DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression.
- De novo transcriptome assembly in chili pepper (Capsicum frutescens) to identify genes involved in the biosynthesis of capsaicinoids.
- Upregulation of the promoter activity of the carrot (Daucus carota) phenylalanine ammonia- lyase gene (DcPAL3) is caused by new members of the transcriptional regulatory proteins, DcERF1 and DcERF2, which bind to the GCC-box homolog and act as an activator to the DcPAL3 promoter.
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- Genome-wide identification and classification of the AP2/EREBP gene family in the cucurbitaceae species.
- Genome-wide identification of AP2/ERF transcription factors in cauliflower and expression profiling of the ERF family under salt and drought stresses..
- Genome-wide identification of the AP2/ERF transcription factor family in pepper (Capsicum annuum L.
- Song X, Li Y, Hou X: Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapa ssp.
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