Literature DB >> 34449796

Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L.

Mona Raeispour Shirazi1, Sara Alsadat Rahpeyma1, Sajad Rashidi Monfared2, Jafar Zolala1, Azadeh Lohrasbi-Nejad1.   

Abstract

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 μM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

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Year:  2021        PMID： 34449796      PMCID： PMC8396717          DOI： 10.1371/journal.pone.0256704

Source DB:  PubMed          Journal:  PLoS One        ISSN： 1932-6203            Impact factor:   3.240


Introduction

Cancer is known as a leading cause of death worldwide, and it is expected that the number of cancer patients will increase to over 22 million cases in the next 20 years [1]. Consequently, the demand for anticancer drugs is overgrowing. Paclitaxel (PC), sold under the trade name Taxol, is a chemotherapy medication used to treat various types of cancer such as ovarian and breast cancers as well as AIDS-related Kaposi’s Scarcorna [2-4]. Compared to other similar compounds, PC’s mode of action against cancer cells is unique in that it inhibits cell proliferation by binding to microtubules. This compound also promotes the stabilization of microtubules at the G2-M phase of the cell cycle [3, 5]. PC is a diterpenoid compound that was first extracted from the western yew tree (Taxus brevifolia). Particularly, hazelnut (Corylus avellana L.) [6], some microorganisms like yew endophytic fungi, and hazelnut endophytic fungi [7, 8] are the other raw resources for PC production. All natural sources produce low levels of PC [9-11]. Although PC can be synthesized through total chemical synthesis, it is found time-consuming, expensive, and low-yielding due to the complexity of the chemical structure of PC [12, 13]. Hence, in response to the increasing demand for the supplies of PC, new alternative approaches are required to be developed. It has been well investigated that Taxus and C. avellana L. cell cultures are promising sources for PC production [4, 14, 15]. Novel strategies were adopted to achieve higher levels of PC production ranging from using elicitors [16, 17], omics studies [18], mathematical modeling [19, 20], and metabolic engineering [21]. Metabolic engineering introduces a rational modification to the genetic makeup of an organism to alter or improve its metabolic profile, and consequently to develop new “non-natural” products. Engineered plant cell lines have the potential to achieve enhanced metabolite production rates [22]. Controlling these complex biosynthetic processes has been provided with the means of understanding the metabolic pathways and advances in molecular biology techniques [23]. However, the amount of PC produced by tissue and cell culture of C. avellana is low. A comprehensive understanding of the PC biosynthetic pathway in hazelnut, particularly the genes encoding rate-limiting enzymes and the enzymes catalyzing these reactions, are influential factors for developments in metabolic engineering and the production of PC to be significantly increased [24, 25]. The biosynthesis pathway of PC contains 19 enzymatic steps starting from the universal diterpenoid precursor called geranylgeranyl diphosphate (GGPP). GGPP is formed through coupling farnesyl diphosphate (FPP) to isopentenyl diphosphate (IPP) by GGPP synthase (GGPPS) [26]. A few PC biosynthetic genes such as GGPPS (Gene Bank Accession No: EF 206343) and CgHMGR (Gene Bank Accession No: EF553534) have been identified in hazelnut [25, 27]. Taxa-4(5), 11(12)-diene is the initial main precursor of the PC biosynthesis pathway. This precursor is catalyzed by taxadien synthase (TS) from GGPP [28]. Taxadien is hydroxylated at the C-5 position and produces taxa-4(20),11(12)-dien-5α-ol [29]. The first acetyltransferase in the PC biosynthetic pathway is taxadien-5α-ol-O-acetyltransferase (T5AT) which converts taxa-4(20),11(12)-dien-5α-ol to taxa-4(20), 11(12)-dien-5α-yl-acetate. Taxa-4(20), 11(12)-dien-5α-ol is acrylated by T5AT at the C-5 position in the presence of acetyl CoA [26]. This reaction is important due to the presence of an essential branch in this position. The gene encoding T5AT with some other genes is involved upstream of the biosynthetic pathway [30, 31]. High expressions of upstream biosynthesis genes such as Taxadien-5α-ol-O-acetyltransferase (TDAT) gene in some tissues or new cultivars of Taxus are assumed as the leading cause for higher yield of PC due to the catalysis of a series of key acetylation and hydroxylation steps [32, 33]. Furthermore, T5AT competes with taxadien-13α-hydroxylase (TαH). TαH like T5AT converts taxa-4(20), 11(12)-diene-5α-ol to taxa-4(20), 11(12) diene 5α, 13α-diol, however, its next steps are unknown and low-expressed. Thus, this branch in the pathway could be targeted for metabolic engineering [34, 35]. These results point to TDAT’s significance in the PC biosynthetic pathway, making it a promising candidate for PC engineering in C. avellana L. In this article, we present the novel information on the isolation and characterization of TDAT from the methyl jasmonate (MeJA)-induced cell suspension of hazelnut, with the goal of better understanding the PC biosynthetic pathway, which could be the subject of future researches.

Materials and methods

In vitro cell culture chemical and biological reagents

In this research, the plant materials were hazelnut (C. avellana L.) cotyledon-derived calli, and cell suspensions. For this purpose, hazelnut seeds were collected from Gilan Province of Iran, (37.1378° N, 50.2836° E). Friable calli and cell suspensions were obtained according to the cells immobilization method [36]. Briefly, the first step was sterilization of the seeds by sodium hypochlorite (5.25%) for 25 min, ethanol 70% (v/v) for two min, sodium hypochlorite (25 min) again, and finally sterile water (3 x 5 min). Almost 1 g from the induced white calli of seed cotyledons in MS medium supplemented with 0.2 mg l-1 6-benzylaminopurine (BA) and 2 mg l-1 2, 4-dichlorophenoxy acid (2.4-D) were suspended in 30 mL liquid media with the same composition. The cell mass obtained from several subcultures in liquid media was immobilized in solid media and produced friable and fast-growing calli. These calli were applied for subsequent experiments and the establishment of the next suspension. Culture medium salts and vitamins were produced from Merck (Kenilworth, New Jersey, USA) pharmaceutical company and other reagents including plant hormones and elicitors were produced from Sigma (St. Louis, Missouri, USA) chemical company.

Elicitor treatment of cell suspension

Frequent subcultured (at least 4 times) and homogenized cell suspensions were used for the elicitor treatment. MeJA stock solution was prepared by dissolving it in 0.1% ethanol and water and then sterilizing it by filtering through 0.22 μm filters. The cell suspension was treated on the seventh day of its cultivation (start of stationary growth phase) with different concentrations of MeJA (0, 50, 100, 150 μM) with three replicates. In the case of control cultures (0 μM MeJA), 10 μL filter-sterilized 0.1% ethanol were added. The elicited cells were harvested after 72 h [17]. Subsequently, the harvested cells were stored in liquid nitrogen for being used in the next molecular studies.

EST assembly strategy for identification of TDAT

The TDAT coding DNA sequence (CDS) was obtained from C. avellana Expressed Sequence Tags (EST) library (https://www.ncbi.nlm.nih.gov/genbank/dbest/). In brief, ESTs were downloaded from several RNA sequence projects (SRA) of hazelnut and used to build up consensus sequences. The EST sequences of TDAT genes were obtained from similar orthologous sequences in other plants utilizing offline BLAST software. These EST sequences were assembled through the “align-then assembles” approach using the Codon Code Aligner V.6.0.2 software (Codon Code Corporation, USA). Then, a TDAT consensus sequence was generated using the assembling operation and then emitting different ESTs. Accordingly, the sequence was cleaned up and a consensus sequence was created using Codon Code Aligner. This sequence was evaluated to find the open reading frame (ORF) and related protein using the ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) was carried out to confirm the ORF and protein of the TDAT gene. Eventually, gene-specific primers were designed for PCR-amplification of the TDAT gene based on bioinformatics analysis. Primer premier (V.6.0) was used to design the primers based on the full ORF of the cDNA consensus sequence (Table 1). Interaction and characteristics of primers were investigated and confirmed with the oligo analyzer (https://www.idtdna.com/pages/tools/oligoanalyzer) V.3.1 program.
Table 1

PCR primers sequences for TDAT gene.

PositionPrimerSequences
Outside ORFForward. 1 5’-GGGGGACAATCTCAAGTTCATT-3’
Outside ORFReverse. 1 5’-GTGGACCAAATTGAACGTACACC-3’
Inside ORFForward. 2 5’-CTGACAATCAGCTCAGAAGGAAAG-3’
Inside ORFReverse. 2 5’-CTAGACCAAGTTGTGACCAGTAG-3’

Extraction of RNA and cDNA synthesis

The total RNA was extracted from MeJA treated hazelnut cells (calli) with a Total RNA isolation kit, DENAZ II ASIA, (cat No.: S-1010, Iran). The concentration and quality of the extracted RNA were analyzed using a Nanodrop spectrophotometer (OneC, Thermo Scientific, USA) and confirmed with agarose gel electrophoresis. Genomic DNA content was removed from the extracted RNA with RNase-free DNAse I (Thermo Scientific (Fermentase), cat no.: ENo521, USA). The first-strand cDNA synthesis reaction was accomplished using a Revert Aid first-strand cDNA synthesis kit (Thermo Scientific (Fermentase), cat no.: K1621, USA).

RT-PCR analysis and gene isolation with nested-PCR

Since gene identification and isolation from cDNA depends on its expression, the cell suspension elicited with MeJA was used for RNA extraction and cDNA synthesis. Different MeJA concentrations were used to induce TDAT expression, and the induced cell mass with 150 μL of MeJA was used for the subsequent RT-PCR and nested-PCR. TDAT gene expression was investigated using reverse transcription PCR (RT-PCR). Nested-PCR was also performed to confirm the characterized ORF. Nested primers were designed in different situations of the consensus sequence. These primers were used to amplify overlapping fragments in order to approve the ORF specification (S1 Fig). The RT-PCR experiment was carried out using Forward.1 and Reverse.1 primers (Table 1). These primers amplified a fragment with a length of 1423 bp and an ORF of 1302 bp. The following reagents were used for RT-PCR and nested-PCR: 1 μL DNase-free water, 1 μL forward primer (10 pmol μL -1), 1 μL reverse primer (10 pmol μL -1), 2 μL cDNA (100–200 ng μL -1), and 10 μL Tag DNA polymerase Mix Red– 1.5 μM Mgcl2 (Ampliqone, cat no.: A150303, Denmark). A volume of 20 μL of each reaction was used for PCR. The RT-PCR program, as well as nested-PCR, were executed as follows: pre-denaturing cDNA at 94°C for 1 min, 34 cycles for denaturing at 94°C for 30 s, annealing at 52± 2°C for 35 s, and 85 s of extension at 72°C. After the last cycle, the amplification was extended at 72°C for 5 min. Annealing at 58°C for 35 s was set for the RT-PCR program with GAPDH primers (S1 Table). PCR purification was done using Ron’s Gel Extraction Kit (BIGRON, cat no.: 802501) for 1423 bp. The cleaned PCR products were subjected to sequencing (Bioneer, South Korea). The nested–PCR amplification and full-length cDNA sequencing were repeated four times.

Bioinformatics analysis and modeling of the T5AT protein

The results of cDNA sequencing were analyzed using Chromas V.1.14 software. Overlapping sequences were edited using the CLC sequencing V.6.1 program, and the final sequence was used for subsequent studies. Protein sequences that identified with more than 70% of the coding region of consensus sequences from different species were selected for sequence alignment and phylogenetic analysis. The relationship between T5AT and proteins downloaded from BLASTp was determined using the web-based Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/), and the maximum likelihood was inferred using the MEGA V.7.0. A Dayhoff model was used to build the phylogenetic construction [37]. To calculate bootstrap (BS) values, 1000 iterations were used. The previously aligned nucleotide sequences of 15 genes were used to construct the evolutionary tree of the TDAT gene, based on the branch-site model, by using the EasyCodeML software, a part of the PAML package [38]. The branches’ lengths and Omega (ω) values were evaluated using Nei & Gojobori method [39] and Bayes Empirical Bayes (BEB) analysis [40].

Secondary structure prediction of the T5AT protein

The secondary structure of the T5AT protein was predicted and assessed using the software. The conserved area of the T5AT gene was identified using the alignment of ortholog sequences for more precision and confidence. InterPro (https://www.ebi.ac.uk/interpro/) was used to represent the domains of T5AT. The PSORT-program (www.genscript.com/tools/wolf-psort) was used to determine the functional location of the T5AT protein. The position of each amino acid in the secondary structure of the protein was determined using the online PORTER (http://distillf.ucd.ie/porter/) and PSIPRED (http://bioinf.cs.ucl.ac.uk/psipred/) programs. The protein storage site and gene ontology of T5AT were performed using PredictNLS (https://predictprotein.org/). This program showed the connection between amino acids and other proteins and their molecular functions. The Signal Peptide program (http://www.cbs.dtu.dk/services/SignalP/) was performed to predict the signal sequence of T5AT. Epestifind software (https://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind) was used to rapidly identify the PEST motif (proline (P), one aspartate (D), glutamate (E), and at least one serine (S), or threonine (T)). ProtParam (https://web.expasy.org/protparam/) and ProtScale (https://web.expasy.org/protscale/) computed various physicochemical properties such as molecular weight, theoretical PI, amino acids composition, instability index, and hydrophobicity scales [41, 42].

T5AT tertiary structure prediction

The tertiary structure of the T5AT protein was assessed using the program described herein. The amino acid sequence associated with the taxadien-5α-ol-O-acetyltransferase enzyme was prepared from the protein sequence database of NCBI with AAB41811 code. The T5AT sequence was compared to the sequences of proteins available in the Protein Database (PDB) to find the appropriate template creating its tertiary structure. The template crystallography file of protein (PDB ID: 4g0b) was obtained from a protein data bank. Modeler software (V.9.20) was used to generate 500 structural models of the T5AT protein. Verify3D and ERRAT compared the similarity of the simulated structure to the template. Procheck and Prosa software was used to evaluate the quality of the space chemistry models. Procheck was used to assess the overall accuracy of the protein structure and the simulated models. For this purpose, parameters such as the quality of the Ramachandran plot were examined [43]. Finally, the similarity of the 3D-structure of the template with the selected model was estimated by measuring the root mean squared deviation (RMSD) using Swiss-Pdb Viewer (SPDBV) software (version 4.1; https://spdbv.vital-it.ch/).

Results

Elicitor treatment of cell suspension and RT-PCR analysis

In the primary studies, no gene (TDAT) expression was observed in the control (non-elicited) calli or cell suspensions (S2 Fig). MeJA elicitor treatments (50, 100, and 150 μM) were then used to induce TDAT gene expression. TDAT gene expression could not be detected at low MeJA concentrations (50 and 100 μM) and it was perceivable at higher concentration of MeJA (S2 Fig). Accordingly, 150 μM MeJA-induced cells were used for RNA extraction and RT-PCR preparation. At this concentration, PCR purification was carried out for 1423 bp and was subjected to sequencing.

Identification and characterization of the TDAT gene with EST assembly and nested-PCR

First, a consensus sequence of TDAT was made of 152 ESTs. The ORFs and related proteins of the consensus sequence were evaluated with ORF finder and confirmed with BLAST(P). Nested-PCR was performed as a supplementary experiment to amplify the full-length cDNA of the TDAT from C. avellana and merely to further confirm the results of the bioinformatics analysis. It amplified the fragments located at the coding regions of TDAT with the designed primers (S3 Fig). The results of nested-PCR were precisely the same as what was expected from the bioinformatics analysis. The cDNA was 1423 bp and, according to ORF finder, it was included in 1302 bp encoding 433 amino acids (S2 Table) (submitted to GeneBank as accession number: TDAT (TAT): MF134435).

Bioinformatics analysis and modeling of T5AT protein

To draw the phylogenetic tree and protected regions, the alignment of amino acids was carried out using Clustal Omega software. The HXXXD motif was very stable and highly conserved. Its amino acid was at positions 164–168 of C. avellana (Fig 1).
Fig 1

Multiple sequence alignment amino acids of TDAT gene with other plants.

The HXXXD motif was indicated with the ▼ symbol above the motif letters.

Multiple sequence alignment amino acids of TDAT gene with other plants.

The HXXXD motif was indicated with the ▼ symbol above the motif letters. The results of phylogenetic analyses suggested that the TDAT gene in C. avellana is more closely related to HHT in Quercus suber and it belongs to the same order as Quercus suber and Juglans regia (Fig 2).
Fig 2

Phylogenetic analysis of identified TDAT of C. avellana with HHT to another plant that had high identity.

One of the statistical parameters to genetically evaluate the evolution process is Omega (ω) value, the rate ratio of nonsynonymous to synonymous substitutions (d/d). ω = 1 suggests neutral expectation; ω< 1 indicates negative (purifying) selection; while ω> 1 shows positive (diversifying) selection [39]. As shown in S4 Fig, the evaluated species are divided into three distinct categories according to the evolution of the TDAT gene. This gene has the same ancestor in Corylus, Quercus, and Juglans. The assessment of nucleotide changes that alter the amino acids (d) relative to nucleotide changes that do not affect the resulting amino acid is a practical and highly efficient method to detect the process of natural selection during gene evolution. According to the matrix obtained from the Nei & Gojobori method, d, d and ω were 0.0838, 0.4804, and 0.1745, respectively, for C. avellana L., indicating a purifying selection for the TDAT gene. Several statistical methods have been developed based on codon substitution models to identify positive selection for a branch in a phylogeny. One of the simulation studies in the phylogenetic analysis by maximum likelihood (PAML), using the maximum-likelihood methods is Bayes Empirical Bayes (BEB) analysis. It has been inferred from the results that Thr106 in C. avellana, had been influenced under positive selection by approximately 98%. The probability of changes in amino acids Gly24, Phe32, Phe34, Pro46, Lys47, Gly50, Ala79, Gly161, Gln206, Val242, Ser307, and Pro376 were between 50% and 79%. The length of the branches showed the number of nucleotide substitutions per codon (S4 Fig). As a result, most nucleotide changes were observed for Juglans (0.27151) in comparison with Corylus (0.26287) and Quercus (0.22245).

Secondary structure prediction of T5AT protein

The results of protein domain analysis, performed using the InterPro database and tools, predicted that the T5AT protein would belong to the family of transferases. The homologous superfamily of the T5AT protein has two domains. The position of the first domain was found to be at amino acids 7–216 and the second domain at 220 to 432. The molecular function of the enzyme was foreseen as the transmission of the acyl group using PredictNLS. The NLS analysis revealed the cytoplasmic localization of this protein. This software confirmed the molecular function predicted by InterPro. All protein binding sites (PBSs) which catalysis the activity and the biological process of this protein in the biosynthesis of the metabolites, were shown by PREDICT NLS (S5 Fig). The PBSs were located at amino acids 1–4, 53–56, 116–120, 212–213, 397–397, and 408–410. Based on the Signal Peptid program, no signal peptide was determined for the T5AT protein. According to Porter’s data prediction, the T5AT protein is formed through the contribution of 29.6% Helix, 26.1% extended or Beta strand, and 44.34% coils (Fig 3). The secondary structure of the T5AT protein was also confirmed with the same result provided by the PSIPRED program with a confidence line having 0 to 9 digits. The PSIPRED prediction of the location of the T5AT protein was performed in 6 positions with different scores. The active site of this protein in the plant cell was predicted to be in the cytoplasm (50%), nucleus (21.5%), mitochondrion (7.1%), peroxisome (7.1%), endoplasmic reticulum (7.1%), and chloroplast (7.1%).
Fig 3

The secondary structure of T5AT protein prediction with PORTER program.

Query-length: 433. H = Helix. E = strand. C = Coil. B = much buried. b = somewhat buried. e = somewhat exposed. E = very exposed.

The secondary structure of T5AT protein prediction with PORTER program.

Query-length: 433. H = Helix. E = strand. C = Coil. B = much buried. b = somewhat buried. e = somewhat exposed. E = very exposed. Epestifind software was used to find the PEST motifs as potential proteolytic cleavage site. Altogether 4 PEST motifs were identified in the T5AT protein located between positions of 1 and 433. A potential PEST motif with 14 amino acids was determined among the positions of 14 to 29, and three poor PEST motifs were considered to be among the amino acid positions of 136–150, 319–337, and 378–398 (S6 Fig). Based on the estimation of Protparam software, the molecular weight of the TDAT protein and its theoretical pI were 48017.62 Da and 6.43, respectively (Table 2). The result of Protscale analysis showed that T5AT had 51 positive electric charges and 53 negative electric charges, due to the presence of lysine/ arginine and aspartate/glutamate. The detailed frequency of amino acids participating in the T5AT structure is provided in Table 2. The molecular formula of this protein was assigned as C2171H3434N560O628S18. The instability index for T5AT was estimated to be 37.94 implying the protein’s stability. The Grand Average of Hydropathicity (GRAVY) value was -0.090. Furthermore, the results of the Kyte & Doolittle plot (Fig 4), as well as the tertiary structure, showed that T5AT protein fragments had 24 hydrophobic regions with amino acids 33–35, 41–47, 64–66, 68–77, 81–83, 87–89, 91–107, 124–132, 135–150, 152–163, 168–176, 189–191, 194–197, 240–243, 264–270, 288–292, 308–317, 328–339, 352–355, 363–373, 378–379, 382–393, 397–405, and 414–429. Pro 142 and Lys 56 exhibited the highest (2,033) and the lowest (-2,644) hydrophobicity residue, respectively.
Table 2

A glance at the prediction of simple physicochemical specifications (amino acids composition) of the T5AT protein.

The amount of amino acid leucine (Leu) in the T5AT protein structure was the most.

Amino acidsnumbersAmount (%)
Ala (a)306.9%
Arg (R)163.7%
Asn (N)122.8%
Asp (D)214.8%
Cys (C)92.1%
Gln (Q)143.2%
Glu (E)327.4%
Gly (G)296.7%
His (H)71.6%
Iis (I)255.8%
Leu (L)429.7%
Lys (K)358.1%
Met (M)92.1%
Phe (F)255.8%
Pro (P)276.2%
Ser (S)276.2%
Thr (T)266.0%
Trp (w)40.9%
Tyr (Y)92.1%
Val (V)347.9%
Pyl (O)00.0%
Sec (U)00.0%

Number of amino acids: 433.

Molecular weight: 48017.62.

Theoretical pI: 6.43.

Fig 4

The results of Kyte and Doolittle diagram about the T5AT protein containing 24 hydrophobic regions.

A glance at the prediction of simple physicochemical specifications (amino acids composition) of the T5AT protein.

The amount of amino acid leucine (Leu) in the T5AT protein structure was the most. Number of amino acids: 433. Molecular weight: 48017.62. Theoretical pI: 6.43.

3D-structure of the T5AT protein

First, similar and most close sequences to the T5AT sequence were obtained using the protein BLAST database. The hydroxycinnamoyl-COA shikimate/quinate hydroxycinnamoyl transferase enzyme, which it’s 3D-structure (PDB code; 4G0B) was identified by Lallemand et al. [44], was considered as a template for the T5AT protein. The sequence identity between the two proteins was determined to be 35%. Five hundred structural models were generated T5AT using Modeller software. Validation tests evaluated the quality of each model, and the model with the highest degree of similarity was considered as the tertiary structure of the T5AT protein which was referred to as MO323. The results of Ramachandran plots (S7 Fig) were summarized in Table 3. Approximately, 87.0% and 88.0% of all amino acids were located within the most favored regions for the template and MO323, respectively. The slight difference between the obtained values can be explained by the position of amino acids in specific regions of the Ramachandran plot, implying that the 3D-structure of MO323 was highly similar to that of the template. The overall quality of the MO323 was checked by comparing Z-scores in Prosa software. The Z-score calculates the total energy of the structure and shows the degree of consistency between the sequence and the tertiary structure of the model [45]. This value was calculated as -9.94 and -8.72 for the template and MO323, respectively. The negative values represented the accuracy of the simulated structure; accordingly, there was a high similarity between MO323 and the template. As shown in Fig 5, the structure of MO323 was located at the position corresponding to the X-ray structures. Thus, the obtained model was sufficiently reliable and was empirically close to the 3D-structure of the pattern. Verify3D calculations showed that 60.51% of the amino acids belonging to MO323 had a score greater than 0.2. This value was 89.46% for the template. This parameter determines the compatibility of the protein 3D-structure with its 1D-structure. Based on the obtained score, the accuracy of the simulated structure and its high quality can be inferred. ERRAT values were determined to be 75.8 and 90.5 for MO323 and the template, respectively. This parameter examines non-bonded interactions between different atom types and the performance rates of 9 amino acids versus other 9 amino acids by statistically comparing with the most similar structures. Based on the results, MO323 was selected as the T5AT 3D-structure from 500 models. The RMSD values for the template and the model (based on backbone and α-carbon) were 0.79 and 0.81 angstroms, respectively. The slight differences between the RMSD values of the two proteins demonstrated their structural similarity. The 3D-structures of the template and MO323 were merged, and the overall similarity and slight differences between them were examined using YASARA software. The results showed that the topology of both proteins’ folding was significantly similar despite the slight differences between the model and the template (Fig 6). Differences were more specified in the turn and coil regions. As shown in Fig 7A, the T5AT enzyme contained 15 beta-strands and 9 alpha-helices in its tertiary structure. Beta-strands were located at the amino acid positions 12–21, 45–51, 30–32, 82–86, 89–95, 100–107, 143–151, 154–163, 231–239, 281–294, 310–318, 368–372, 388–392, 399–405, and 412–420. Alpha-helices were located at amino acid positions with 35–40, 61–72, 168–184, 240–249, 261–278, 319–326, 328–341, 343–355, and 423–432. The T5AT enzyme belongs to the transferase superfamily which is commonly found in plants and fungi. The HXXXD motif makes up part of the active site of all enzymes in this superfamily [46]. This motif located at positions 164–168 of the T5AT protein structure contains the His-Cys-Leu-His-Asp amino acid sequence forming a turn region between the eighth beta-strand and the tertiary alpha-helix (Fig 7B).
Table 3

Related parameters for confirmation of the simulated structure and its similarity to the pattern.

ModelProcheckVerify 3DErrat
Most favored regionsAdditional allowed regionsGenerously allowed regionsDisallowed regions
4G0B 87.0%12.7%0.3%0.0%89.46%90.3061
MO323 88.8%10.4%0.8%0.0%60.51%75.5882
Fig 5

Z-score values for (A) the template, (B) the simulated model (MO323). Both structural models are located in the position of X-ray structures.

Fig 6

Comparison of the surface of the 3D-structure belongs to the template and MO323 (red and yellow, respectively), differences of structures are relative to turn and coil regions.

Fig 7

3D-structure of the T5AT enzyme presented by YASARA program.

(A) Beta-strands and alpha-helixes are yellow and blue, respectively. This model has 15 beta-strands and 9 alpha-helices; (B) HXXXD motif in T5AT enzyme contains His 164, Cys 165, Leu 166, His 167 and Asp168.

Z-score values for (A) the template, (B) the simulated model (MO323). Both structural models are located in the position of X-ray structures.

3D-structure of the T5AT enzyme presented by YASARA program.

(A) Beta-strands and alpha-helixes are yellow and blue, respectively. This model has 15 beta-strands and 9 alpha-helices; (B) HXXXD motif in T5AT enzyme contains His 164, Cys 165, Leu 166, His 167 and Asp168.

Discussion

A considerable number of research papers have been devoted to investigating the taxol biosynthetic pathway in Taxus and its producing microorganisms [29, 47]. Despite all these efforts, that pathway still has ambiguities in details and it is yet unknown in hazelnut except for the early two genes [25, 27]. The key acetylation step by taxadien-5α-ol-O-acetyltransferase (TDAT or TAT), as the early bottleneck in the pathway, has been highlighted [48]. Moreover, the key genes participating in the next hydroxylation steps, are affected by the TDAT catalytic step [32]. In this study, TDAT ortholog was detected and identified in C. avellana L. using the experimental and in-silico analysis. C. avellana is known as a candidate source for PC production [49]. The full-length cDNA encoding TDAT from C. avellana (gene accession number: MF134435) was 1423 bp, similar in size to omega-hydroxypalmitate o-feruloyl transferase (HHT). The cDNA of TDAT contains a 1302 bp ORF encoding a protein of 433 amino acids. The constructed phylogenetic tree indicated a high degree of identity between TDAT and HHT. Corylus, Quercus suber, Juglans regia, Morus notabilis, and Ziziphus jujube resided on the same branch of the phylogenetic tree. It should be noted that each of these plants belongs to different families, indicating this gene was protected in distant families. Early clarifying assessments revealed that TDAT is also multi-specific in Taxus [48, 50]. Investigation on the TADT genetic evolution showed that Thr106 had a positive change of 98% compared to other species. The coding sequence of this amino acid for Corylus is ACA, while other species have GAA (12 cases) and GAG (2 cases) for Glu at this position. Thr106 was positioned in the beta structure of the T5AT 3D-structure and away from the enzyme active site. Therefor altering this amino acid probably has little effect on the active site. Analysis of the enzyme 3D-structure showed that Gly24, Phe32, Phe34, Pro46, Lys47, Ala79, Gly161, Ser307, and Pro376 are close to the active site, and altering of these amino acids might affect their function. The taxadien-5α-ol-O-acetyltransferase gene has been identified in the yew, another main source of PC. This gene, called TmTAT, was known in T. media. The ORF of the TmTAT gene was 1317 bp encoding 439 amino acids [51]. The molecular weight of the T5AT protein was 48.17 kDa. The theoretical pI of T5AT was 6.43, and the instability index for the protein was 37.94. The result of BLASTp indicated that the T5AT protein was closely similar to the HHT protein in Juglans regia and Ziziphus jujube. The isoelectric point of HHT in J. regia and Z. jujube was 5.83 and 5.66, respectively. The instability index of HHT in J. regia and Z. jujube was calculated as 39.31 and 35.75, respectively. The molecular weight of the HHT protein was very close to that of the T5AT protein—approximately 48 kDa. The HHT protein consisted of 432 amino acids in both plants. To investigate the evolutionary relations between the transferase in Taxus and Corylus, a phylogenetic tree was constructed based on the amino acid sequences of TmTAT and transferase from other Taxus species. The results highlighted that TmTATs, (T5ATs) from T. chinesis and T. cuspidata were grouped into the cluster with the shortest distance from the T5AT of C. avellana while the TmTAT from T. canadensis was categorized into a cluster farther away from C. avellana. Moreover, the T5ATs from C. avellana and Taxus species were found to be dissimilar [24]. The taxadien-5a-ol-O-acetyl transferase ORF in T. chinensis had 1275 nucleotides that encodes 425 amino acids. The protein had an isoelectric point of 5.39 and a molecular weight of 47 kDa [52]. The molecular weight of the TmTAT was 49 kDa, and its instability index was 35, similar to the other Taxus species. On close scrutiny, TmTAT was strongly expressed in the leaves, weakly expressed in the stems, and had no expression in the fruits of T. media. A detailed look at this tissue expression pattern revealed that this is a tissue-specific gene [24]. The T5AT protein had two domains as did the HHT protein in Quercus suber. While, the position of the first domains of both proteins were located at amino acid positions 7–216, but the second domains were found at different positions. In contrast, the second domain of HHT was located at amino acid positions 221–431, which differed in the position of just one amino acid in T5AT (220 to 430). However, these second domains indicated that T5AT and HHT belong to the superfamily of transferase enzymes [24, 52]. The T5AT and HHT proteins, as well as most acetyltransferase enzymes, have a functional HXXXD motif. This motif resided in amino acid position 164 [46]. In T. chinensis, this protein has a functional HXXXD motif with the function of transferring acetyl from the precursor of the CoA-acetyl and additionally, two other domains at positions 7–214 and 223–431 [52]. Based on the NCBI database, the TmTAT protein has a second domain belonging to the family of the transferase, a functional HXXXD motif, which plays a role in the transmission of the acetyl group, and also two domains located at amino acid positions 8–214 and 223–431 [24]. It was revealed that the HXXXD motif is probably part of the active site [46]. The previous studies reported a lack of signal peptide in T5AT, HHT, and TmTAT proteins; therefore, these proteins probably act in cytoplasm. The biological process of the T5AT protein was predicated in the biosynthesis of metabolites, transferase activity, and the transmission of acetyl group. The molecular function of taxadien-5a-ol-O-acetyltransferase (T5AT) was identified as transmitting acetyl group from acetyl-CoA and producing the T5AT enzyme in T. cuspidate [34]. Furthermore, the molecular function and biological process of the TmTAT enzyme have been reported to be the same as those of T5AT in T. cuspidate [24]. In this study, 29.6% Helix, 26.1% Extended, and 44.34% Coils were observed in the secondary structure of T5AT with 433 amino acids, while 37% Helix, 26% Beta strand or Extended, and 37% Coils contributed to the formation of the TmTAT protein [52]. The pattern of the T5AT protein in drawing the three-dimensional structure was HOST in Coffea canephora (PDB Id: 4g0b). It was confirmed that His-153 in HCT is an active site, as the His-153-ALa mutation had no enzymatic activity as a catalytic enzyme. The His-153 in HCT from the conserved HXXXD motif was confirmed as a catalytic residue by mutagenesis [53]. Therefore, His-164 is found in the active site of the T5AT protein (C. avellana). The active sites in the T5AT in T. cuspidate were also included His-164 and Asp-373. As a result, it is most likely that Asp-381 is another amino acid in the active site involved in the T5AT (C. avellana). ALa-156-Ser mutants produced 4-fold more diCQAs. Ser is a polar amino acid with an OH group. Since His is a polar amino acid, it is expected that His-167 will improve enzyme activity [44]. The HST protein pattern in Arabidopsis thaliana (PDB Id: 5kjs) was used to draw the tertiary structure of the HCT protein in C. canephora. HCT and HST proteins are located in the BAHD acyltransferase family. This protein family is recognized by the sequence homology and HXXXD and DFGWG conserved motifs and considered as the main enzymes in a wide range of secondary metabolites’ biosynthesis pathway with a wide variety of substrates. In the active site, the HHAAD and DFGWG contain the two essential amino acids His153 and Asp380. [26, 44]. Our findings show that these two motifs are found in the HCT structure in the regions 157–157 and 381–385, with the same sequence. The first motif, HCLHD, is found at position 164–168 in the T5TAT structure sequence, and the second motif, which has a completely identical amino acid sequence to HCT and HST, is found at position 381–385 in the protein. The existence of the conserved motifs in the structure of T5TAT protein indicates that it belongs to the BAHD acyltransferase family and transferase superfamily.

Conclusion

Bioinformatics research was used to identify and characterize taxadien-5-ol-O-acetyltransferase (TDAT), which catalyzed the third stage of PC biosynthesis in C. avellana. Our success in finding this gene was due to the use of elicitor to induce gene expression, as well as the use of the EST library and bioinformatics studies. It can be assumed that the taxadien-5α-ol-O-acetyltransferase enzyme has undergone numerous changes during its evolution. Although this enzyme varies significantly from Taxus’ T5AT enzyme, their active site, and conserved motif are identical. It also has similar performances in different plants. This gene locates in the early steps of the PC biosynthetic pathway. On their previous substrate, taxadien-5-ol, T5AT competed with T13Ha. As a result of its location at the start of the pathway and on the branch, this gene is crucial. This is a very low-expression gene that necessitates the use of high-level elicitors for its identification. Herein, for the first time, the full length and features of the TDAT were identified using bioinformatics analysis rather than costly, time–consuming, and labor-intensive work. Understanding the sequence and characteristics of TDAT will offer promisingly to PC engineering programs and maximize its production in hazelnut for future studies.

The position of full-length cDNA and nested-PCR primers containing TDAT gene.

The arrows define the primers and the green rectangles illustrate nested-PCR products for each primer. (DOCX) Click here for additional data file.

Agarose (1%) gel electrophoresis.

Evaluation of RT-PCR product of the different concentrations of MeJA induced cells and PCR purification. L: (ladder 100 bp), 1: Negative control (water), 2: RT-PCR products without any treatment (control), 3: RT-PCR product with 50 μM of MeJA, 4: RT-PCR product with 100 μM of MeJA, 5: RT-PCR product with 150 μM MeJA, 6: Negative control, 7: PCR product after PCR-purification in 150 μM of MeJA. The grouping of gels which have been cropped from different gels was identified with vertical white lines. (DOCX) Click here for additional data file.

Agarose (1%) gel electrophoresis of nested-PCR analysis of C. avellana; Ladder (L) 100bp,1: Forward 1 + Reverse 1 (Fr1+Rr1), 2.

Forward 1+ Reverse 2 (Fr1+Rr2), 3. Forward 2 + Reverse 1 (Fr2+Rr1), 4. Forward 2 + Reverse 2 (Fr2+Rr2), 5. Positive control (GAPDH primers), 6. Negative control (water). (DOCX) Click here for additional data file.

Evolutionary relationships of C. avellana L. TDAT gene with 15 geneses, based on the branch-site model, by using the EasyCodeML software (PAML package).

(DOCX) Click here for additional data file.

The protein binding sites (PBS) showed by PREDICT NLS.

All indicated parts with red diamonds are PBS. The regions are 1–4, 53–56, 116–120, 212–213, 397–397, and 408–410. (DOCX) Click here for additional data file.

The position of the PEST motif was detected with Epestifind software.

(DOCX) Click here for additional data file.

The Ramachandran plot and the frequency of amino acids in a different position for C. avellana L.

(A) 4G0B template (B) MO323. In the picture, [A, B, L] show a very favored region for amino acids. [a, b, l, p] show the allowed zone, and [~a, ~b, ~l, ~p] show the permitted regions with ignorance. (DOCX) Click here for additional data file.

PCR primers sequences for GAPCH gene.

(Thermo Scientific (Fermentase), cat no.: K1621, USA). (DOCX) Click here for additional data file.

The results of ORFs, online software.

The most portable framework and the tallest of ORF are in + 1 and the nucleotide of 97 to 1398. (DOCX) Click here for additional data file. (PDF) Click here for additional data file. 22 Apr 2021 PONE-D-21-05459 ​Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L. PLOS ONE Dear Dr. Rahpeyma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jun 06 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Nevertheless it needs some serious improvements in the literature review as well as how the data and figures are explained and presented. Some figures are blurry and couldn’t be assessed properly. It needs to compare more with recents studies where the TAT has been utilised and highlight more the relevance of the work too. Please do find the specific comments on the pdf file attached Reviewer #2: Abstract Line no. 16 – 18: ‘and tissue cultures of Taxus 16 species and’ not correlating here. Reframe the sentence. Line no. 20: change as ‘this study was undertaken to …..’ Introduction Line no. 44 – 46: Sentence starting as ‘In particular……’ rewrite the sentence Line no. 55 – 58: Sentence starting as ‘The deep and detailed understanding’ rewrite the sentence. Line no. 65: Change as ‘The initial main precursor of PC’ Materials and Methods Line no. 88: ‘were procured from Merck’ Line no. 89: were procured from Sigma’ Line no 94: dissolving Results Line no. 195: (non-elicited) Line no. 196 and 197: Remove (0) Line no. 200: change as ‘for 1423 bp and were subjected to sequencing. Line mo. 305 – 308: consider rewriting the sentence General Comments The authors have to improve the language in the entire manuscript to meet the journal standard. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. 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Submitted filename: PONE-D-21-05459_reviewer.pdf Click here for additional data file. 2 Jun 2021 Dear Editor-in-Chief, We would like to deeply thank the editorial office and the reviewers for the constructive comments which helped us to improve the paper and thank you for giving us the opportunity to submit a revised draft of our manuscript titled "Identification and in-silico Characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) Gene in Corylus avellana L". The authors tried to address all reviewers’ comments in the revised manuscript. We hope our revision has improved the paper to a level of their satisfaction. Number-wise Responses to the specific editorial comments, suggestions, and queries are given below; the revisions in the manuscript have been marked highlighted with track changes in a file labeled ‘ Revised manuscript with track changes. Enclosed, please find the revised version of the manuscript entitled: “Identification and in-silico Characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) Gene in Corylus avellana L. By: Mona Raeispour Shirazi, Sara Alsadat Rahpeyma, Sajad Rashidi Monfared, Jafar Zolala and Azadeh Lohrasbi-Nejad1 Sincerely yours, Dr. Sara Alsadat Rahpeyma Assistant Professor of Plant Breeding Department of Agricultural Biotechnology Faculty of Agriculture Shahid Bahonar University of Kerman Iran Responses to journal requirements: - The authors tried to improve and adjust the manuscript style to meet PLOS ONE’s style found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. Track changes were used to mark the changes, including text format, figures, tables, references, grammatical adjustments, etc, across the updated manuscript. - - Two gel figures are included in the manuscript "PONE-D-21-05459" (Fig 2. and S1 Fig). The authors attempted to compile original gel figs in a single PDF file format called "S1-row pictures," which contains all the original and unadjusted gel images, included in the manuscript’s main figures and supplemental figures. Fig 2. is in the public data presented in the main article and S1 Fig is in the ‘supporting information’ section. Also, the author tried to consider all requirements for gel reporting, such as labeling and annotating the experimental and not included lanes, molecular markers’ weight, and so on. Unfortunately, we lost all of the original gel images due to a serious problem with my student (Mona Raeispour) laptop, and we only have cropped images retrieved from emails. But I, as the corresponding author, ensure the editorial board that no manipulating has been done in figures. The cropped lanes of images were identified with the blanks among the parts. The photos were captured using the VIBER Company's Quantum gel documentation imaging technology. The authors hope that the revision will meet PLOS ONE's approval. Response to Comments by Reviewer 1 We'd like to express our gratitude to the Reviewer for his or her constructive comments and suggestions. Based on his/her remarks, various revisions were made to improve the quality of the manuscript. The modifications were highlighted with track changes through the revised manuscript. The next sections contain our responses to the dear reviewer comments Comment No.1: This manuscript presents interesting bioinformatic findings about TAT in hazelnuts species, which will be interesting to the community, specially the prediction of secondary and tertiary structures. Nevertheless it needs some serious improvements in the literature review as well as how the data and figures are explained and presented. Response: Thanks to the reviewer for the kind comment on the manuscript. The author tried to improve the literature review by including more recent studies and providing additional details about the presented study. The modifications have been addressed in the text as well as in the separate responses to the attached comments. Comment No.2: Some figures are blurry and couldn’t be assessed properly. Response: We considered the reviewer’s suggestion and Figs 1, 2, 3, 6, and 9 changed to more appropriate ones. All figures were checked to meet journal style. Comment No.3: It needs to compare more with recents studies where the TAT has been utilised and highlight more the relevance of the work too. Response: To highlight the significance of the subject, the recent and relevant TAT studies were added to the text throughout the introduction and discussion sections. They are addressed via track changes. The following new references were added to the introduction and discussion: - Salehi M, Moieni A, Safaie N, Farhadi S. Whole fungal elicitors boost paclitaxel biosynthesis induction in Corylus avellana cell culture. Plos one. 2020;15(7):e0236191. - Yu C, Zhang C, Xu X, Huang J, Chen Y, Luo X, et al. Omic analysis of the endangered Taxaceae species Pseudotaxus chienii revealed the differences in taxol biosynthesis pathway between Pseudotaxus and Taxus yunnanensis trees. BMC plant biology. 2021;21(1):1-13. - Salehi M, Farhadi S, Moieni A, Safaie N, Hesami M. A hybrid model based on general regression neural network and fruit fly optimization algorithm for forecasting and optimizing paclitaxel biosynthesis in Corylus avellana cell culture. Plant Methods. 2021;17(1):1-13. - Farhadi S, Salehi M, Moieni A, Safaie N, Sabet MS. Modeling of paclitaxel biosynthesis elicitation in Corylus avellana cell culture using adaptive neuro-fuzzy inference system-genetic algorithm (ANFIS-GA) and multiple regression methods. PloS one. 2020;15(8):e0237478. - Sanchez-Muñoz R, Bonfill M, Cusidó RM, Palazón J, Moyano E. Advances in the regulation of in vitro paclitaxel production: Methylation of a Y-Patch promoter region alters BAPT gene expression in Taxus cell cultures. Plant and Cell Physiology. 2018;59(11):2255-67. - Sanchez-Muñoz R, Perez-Mata E, Almagro L, Cusido RM, Bonfill M, Palazon J, et al. A novel hydroxylation step in the taxane biosynthetic pathway: a new approach to paclitaxel production by synthetic biology. Frontiers in Bioengineering and Biotechnology. 2020;8:410. - Kuang X, Sun S, Wei J, Li Y, Sun C. Iso-Seq analysis of the Taxus cuspidata transcriptome reveals the complexity of Taxol biosynthesis. BMC plant biology. 2019;19(1):1-16. - Kanda Y, Nakamura H, Umemiya S, Puthukanoori RK, Murthy Appala VR, Gaddamanugu GK, et al. Two-Phase Synthesis of Taxol. Journal of the American Chemical Society. 2020;142(23):10526-33. doi: 10.1021/jacs.0c03592. - Wang T, Chen Y, Zhuang W, Zhang F, Shu X, Wang Z, et al. Transcriptome sequencing reveals regulatory mechanisms of taxol synthesis in Taxus wallichiana var. Mairei. International journal of genomics. 2019. - He C-T, Li Z-L, Zhou Q, Shen C, Huang Y-Y, Mubeen S, et al. Transcriptome profiling reveals specific patterns of paclitaxel synthesis in a new Taxus yunnanensis cultivar. Plant Physiology and Biochemistry. 2018;122:10-8. - Zhou X, Zhu H, Liu L, Lin J, Tang K. A review: recent advances and future prospects of taxol-producing endophytic fungi. Applied Microbiology and Biotechnology. 2010;86(6):1707-17. doi: 10.1007/s00253-010-2546-y. - Walls LE, Malcı K, Nowrouzi B, Li RA, d'Espaux L, Wong J, et al. Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies. Biotechnology and Bioengineering. 2021;118(1):279-93. - Walker K, Ketchum RE, Hezari M, Gatfield D, Goleniowski M, Barthol A, et al. Partial Purification and Characterization of Acetyl Coenzyme A: Taxa-4 (20), 11 (12)-dien-5α-olO-Acetyl Transferase That Catalyzes the First Acylation Step of Taxol Biosynthesis. Archives of biochemistry and biophysics. 1999;364(2):273-9. The specific comments on the pdf file attached Abstract Comment No.1: please clarify that T5AT is encoded by TDAT gene. Response: Thank you for making the suggestion. The phrase “, encoded by TDAT,” was added to the text. Comment No.2: The abstract should be improved with a nice conclusion. Response: The author tried to create a more appropriate conclusion for the abstract. Introduction: Comment No.3: In general introduction is good but the novelty and the aim of study are poorly written. Therefore, introduction should be improved with a better conclusion. Also, the current studies on TAT characterization or production could be mentioned here. Response: Thanks to the reviewer for the comment. The introduction now includes recent studies on TDAT and taxol that are relevant to the issue. A better conclusion has also been added to the introduction. For this aim the sentences “These results point to TDAT's significance in the PC biosynthetic pathway, making it a promising candidate for PC engineering in C. avellana L. In this article, we present novel information on the isolation and characterization of TDAT from methyl jasmonate (MeJA)-induced hazelnut cell suspension, with the goal of better understanding the PC biosynthetic pathway, which could be the subject of future research.” was added to the introduction. Materials and methods: Comment No.4: should not be "bought from"? Response: The sentence “Culture medium salts and vitamins were provided from Merck” was replaced with “Culture medium salts and vitamins were produced from Merck” Comment No.5: the same Response: The modification is like the previous response. Comment No.6: the link does not work, please provide the correct one Response: The link was replaced with https://www.idtdna.com/pages/tools/oligoanalyzer. The other links within the text were checked and updated. Comment No.7: this section needs corrections and should be rewritten Response: The author tried to improve the section and rewrite some sentences to get more clarified. Comment No.8: Please clarify here. Was qPCR used for gene expression detection? RT-PCR stands for reverse transcription PCR. Response: In this study, qRT-PCR was not used. This part was clarified with the sentence “TDAT gene expression was investigated using reverse transcription PCR (RT-PCR). Comment No.9: should be reworded Response: The sentence was changed to “These primers were used to amplify overlap fragments in order to approve the ORF specification” Comment No.10: The primer names should be same as the table Response: The name of primers replaced with “forward. 1 and reverse. 1” the same as other places within the text. Comment No.11: Fig S1 should be mentioned here Response: The modifications have been considered. Comment No.12: The figure caption should be reworded Response: The modifications have been considered and “the situation of cDNA full length and nested-PCR primers comprising TDAT gene.” was replaced with “The position of full-length cDNA and nested-PCR primers containing TDAT gene”. Comment No.13: The links given should be corrected, none of them is working. Some of them are even not in a correct web-link format. Response: All the links in the text have been checked and corrected. Comment No.14: part a and part b should be merged in a single figure as Fig. 2 Response: The modifications have been considered. Comment No.15: if possible, a better image could be used Response: The authors have used their best image. So Fig 2 only was merged into a single image and the resolution was improved in his section. Comment No.16: italic Response: The modifications have been considered. Comment No.17: the frame on this motif should be replaced or the letters can be differently colored to emphasize Response: According to the reviewer's comment, the frame was replaced with the ▼ symbol above the motif letters, in this figure. A few errors in the typography of motif HXXXD were also corrected inside the text. Comment No.18: lowercase Response: The modifications have been considered Comment No.19: Please correct the software name which should be PredictNLS Response: Thanks to the reviewer for the comment. The name of the software was correct with PredictNLS. Comment No.20: make sure this figure is legible Response: The author attempted to replace the figure with a more legible one. Comment No.21: part a and b should be merged into a single figure Response: Thanks to the reviewer for the comment. Two separate figures, which are the outputs of the software, were designed into one figure. According to similar references (e.g., Safavi et al., 2019; Elengoe et al., 2014; pandey et al., 2014), parts A and B do not merge into a single image. So, two parts are labeled A and B in this figure, but if the respected reviewer suggests it, the author will merge the two parts in the next revision. - Safavi, A., Kefayat, A., Sotoodehnejadnematalahi, F. et al. In Silico Analysis of Synaptonemal Complex Protein 1 (SYCP1) and Acrosin Binding Protein (ACRBP) Antigens to Design Novel Multiepitope Peptide Cancer Vaccine Against Breast Cancer. Int J Pept Res Ther 25, 1343–1359 (2019). https://doi.org/10.1007/s10989-018-9780-z -Elengoe, A.; Naser, M.A.; Hamdan, S. Modeling and Docking Studies on Novel Mutants (K71L and T204V) of the ATPase Domain of Human Heat Shock 70 kDa Protein 1. Int. J. Mol. Sci. 2014, 15, 6797-6814. https://doi.org/10.3390/ijms15046797 -Pandey, Saurabh & Negi, Yogesh & Reddy, Subramanyam & S, Krishna Murthy & Arora, Sandeep & Kaul, Dr. Tanushri. (2014). Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance. Bioinformation. 10. 119-123. 10.6026/97320630010119. Discussion: Comment No.22: discussion should be improved. the most parts look like a repetition of the results section. the importance and potentials of the findings/results should be interpreted and elaborated. the significance of elucidation of the sequence and the structure of this enzyme should be clearly indicated. the recent studies about this enzyme (characterization or heterologous expression in microbial hosts) should be mentioned here and the potential of this study should be discussed. please see below for other notes. Response: Thanks to the reviewer for the suggestion. The author has been tried to improve the discussion part by adding some new complementary references and rewriting some sentences and also, adding some new sentences to make more explanations and clarifications. Comment No.23: in contrast, Response: The modification “In contrast” has been done. Comment No.24: should be reworded or removed Response: the sentence “this process was the biological process in the pc biosynthesis” was removed. Comment No.25: a single aa cannot be an active site. rather, it should be in active site Response: Thanks for the reviewer comment. Based on the suggestion, the authors changed the sentences and, as a result, the subsequent related sentences. the modified sentences: “Therefore, His-164 is found in the active site of the T5AT protein (C. avellana). The active sites in the T5AT in T. cuspidate were also included His-164 and Asp-373. As a result, it is most likely that Asp-381 is another amino acid in the active site involved in the T5AT (C. avellana).” Comment No.26: activity site is not a correct term. should be active site. this term should be corrected in all parts of the text Response: The “activity site” has been replaced with the “active site” in the whole text. Comment No.27: this part should be improved too Response: The modification was made according to the suggestion. Comment No.28: conclusion is poorly written, should be improved Response: The author tried to improve the conclusion by replacing and rewriting some sentences. Comment No.29: not a clear statement, should be improved/clarified Response: The sentence “It can be deduced that the taxadien-5α-ol-O-acetyltransferase enzyme has undergone many changes during development.” was replaced with “It can be assumed that the taxadien-5α-ol-O-acetyltransferase enzyme has undergone numerous changes during its evolution”. Comment No.30: This gene was in the primary PC biosynthetic pathway. Response: the mentioned sentence was removed. Comment No.27: Please correct the underlined parts in fig 1 Response: Fig 1 was modified according to the suggestion. Response to Comments by Reviewer# 2 We would like to thank the Reviewer for his/her constructive comments and suggestions. Based on his/her remarks, various revisions have been made to improve the quality of the manuscript. Our responses to the review comments are presented next. Abstract Comment No.1: Line no. 16 – 18: ‘and tissue cultures of Taxus 16 species and’ not correlating here. Reframe the sentence. Response: Thanks for the reviewer comment. According to the suggestion, the authors improved the sentence with “Natural materials including tissue cultures of Taxus species and, most recently, hazelnut (Corylus avellana L.) represent the most promising sources of this compound.” Comment No.2. Line no. 20: change as ‘this study was undertaken to ….. Response: The sentence “this study was undertaken to…” has been replaced with the “this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut.” Introduction Comment No.3: Line no. 44 – 46: Sentence starting as ‘In particular……’ rewrite the sentence Response: the mentioned sentence was improved as follows: “Particularly, hazelnut (Corylus avellana L.) [6], some microorganisms like yew endophytic fungi, and hazelnut endophytic fungi [7, 8] are the other raw resources of the PC production.” Comment No.4: Line no. 55 – 58: Sentence starting as ‘The deep and detailed understanding’ rewrite the sentence. Response: The mentioned sentence was replaced with “A comprehensive understanding of the PC biosynthetic pathway in hazelnut, particularly the genes encoding rate-limiting enzymes and the enzymes catalyzing these reactions, are influential factors for developments in metabolic engineering and and for the production of PC to be significantly increased” Comment No.5: Line no. 65: Change as ‘The initial main precursor of PC’ Response: The sentence was modified according to the comment. Materials and methods Comment 6: Line no. 88: ‘were procured from Merck’ Response: The modification was considered Comment 7: Line no. 89: ‘were procured from Merck’ Response: The modification was considered Comment 8: Line no 94: dissolving Response: The modification was considered Results Comment 9: Line no. 195: (non-elicited) Response: The modification was considered Comment 10: Line no. 196 and 197: Remove (0) Response: The modification was considered Comment 11: Line no. 200: change as ‘for 1423 bp and were subjected to sequencing. Response: The modification was considered Comment 12: Line mo. 305 – 308: consider rewriting the sentence Response: The sentence was modified as follows: “Primarily, similar and most close sequences to the T5AT sequence were obtained using the protein BLAST database. The hydroxycinnamoyl-COA shikimate/quinate hydroxycinnamoyl transferase enzyme, which it’s 3D-structure (PDB code; 4G0B) was identified by Lallemand et al. [44], was considered as a template for the T5AT protein.” General comment: Comment 13: The authors have to improve the language in the entire manuscript to meet the journal standard. Response: Thank you very much to the reviewer. The author attempted to proofread and refine the entire manuscript's grammar to meet the journal’s standard. The grammatical revisions have been marked with track changes inside the text. Best Regards 6- PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Response: No. Submitted filename: responses to reviewer.docx Click here for additional data file. 25 Jun 2021 PONE-D-21-05459R1 ​Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L. PLOS ONE Dear Dr. Rahpeyma, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Balamurugan Srinivasan Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: N/A ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: (No Response) ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) Reviewer #3: The submitted manuscript by Shirazi et al., shows the identification of TDAT gene and provides interesting in silico analyses of the TDAT gene. The data will be interesting to the readership; specifically, the community works on PC biosynthetic pathway. However, the paper needs revision, particularly in terms of gene identification and the methodology adopted for the study. The authors shall rephrase all the RT-PCR into sqRT-PCR, which misinterpret that the authors had performed real-time quantitative PCR. Based on figure 2, all lanes 2,3 and 4 showed the smear, and, intriguingly, only the 150 uM resulted in the amplification of the TDAT gene. What is the dark line present in figure 2 between lane 3 and lane 4? The authors claimed that gel cropped from different gels was differentiated by white lines; however, no such differentiation exists in this figure. The authors claimed that they had purified the PCR product obtained from lane 5. Was it gel elution or purification? P7 L155- authors mentioned as gel extraction- these details may be precisely given in the results section. Moreover, lane 7 does not seem to contain the purified products, instead, it looks more smear than the unpurified products (Lane 5). The authors explain this context. Based on figure 2, the presence of TDAT amplicon in the lanes 2, 3 an 4 can not be ruled out. There might be some problem with the purification of PCR amplicons from the gel. Collectively, to avoid all these misperceptions, the authors are highly recommended to perform real-time quantitative PCR analysis to detect the relative mRNA level of TDAT gene at 0, 50, 100 and 150 uM of MeJa. The number of figures may be reduced. Few figures may be shifted to the Supplementary file. For instance, Figs 1, 2, 3, 5, 7 may be moved to supplementary file. Minor points: P4 L96: What is the content/quantity of callus taken to generate suspension culture as per the given protocol, which may be provided in the methods section. P4 L100: Which term the authors imply: produced or procured? P6 L150: µm P6 L 154: Primer sequences may be given in the table P19 L454-455: This claim warrants the use of qPCR analysis, considering the inconsistent result of the given sqRTPCR gel pictures. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: Yes: Muthukrishnan Arun Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 7 Aug 2021 Dear Editor-in-Chief, We would like to deeply thank the editorial office and the reviewers for the constructive comments which helped us to improve the paper and thank you for giving us the opportunity to submit a revised draft of our manuscript titled "Identification and in-silico Characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) Gene in Corylus avellana L". The authors tried to address all reviewers’ comments in the revised manuscript. We hope our revision has improved the paper to a level of their satisfaction. The revisions in the manuscript have been marked highlighted with track changes in a file labeled ‘Revised manuscript with track changes. Enclosed, please find the revised version of the manuscript entitled: “Identification and in-silico Characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) Gene in Corylus avellana L.” By: Mona Raeispour Shirazi, Sara Alsadat Rahpeyma, Sajad Rashidi Monfared, Jafar Zolala and Azadeh Lohrasbi-Nejad1 Sincerely yours, Dr. Sara Alsadat Rahpeyma Assistant Professor of Plant Breeding Department of Agricultural Biotechnology Faculty of Agriculture Shahid Bahonar University of Kerman Iran Response to Comments by Reviewer 3 We'd like to express our gratitude to the Reviewer for his or her constructive comments and suggestions. Based on his/her remarks, various revisions were made to improve the quality of the manuscript. The modifications were highlighted with track changes through the revised manuscript. The next sections contain our responses to the dear reviewer comments. Comment No.1: The authors shall rephrase all the RT-PCR into sqRT-PCR, which misinterpret that the authors had performed real-time quantitative PCR. Response: Much appreciated to the reviewer for the kind comments on the manuscript. In this study, the novel EST assembly strategy has been applied for identification of TDAT gene, and subsequently reverse transcription (RT)-PCR technique was developed to isolate TDAT gene from cDNA. As you know, the term “RT-PCR” is used for amplification, and semi-quantitative reverse transcription (sqRT-PCR) is a simple and specific method for relative quantitative RNA and amplification and determination of the quantity of PCR products. Since in this paper, the quantity measure was not intended, so it seems that RT-PCR is a more reasonable term in this case. It’s also worth to noting that the term “RT-PCR” was use in different studies (e.g. - Molecular cloning and expression analysis of glutathione reductase gene in Chlamydomonas sp. ICE-L from Antarctica, Marine Genomics, Volume 5, 2012. - Dongmei Yin, Dian Ni, Lili Song, Zhiguo Zhang, Isolation of an alcohol dehydrogenase cDNA from and characterization of its expression in chrysanthemum under waterlogging, Plant Science, Volume 212, 2013. - Chun-Hong Li, Yong-Qing Zhu, Yu-Ling Meng, Jia-Wei Wang, Ke-Xiang Xu, Tian-Zhen Zhang, Xiao-Ya Chen, Isolation of genes preferentially expressed in cotton fibers by cDNA filter arrays and RT-PCR, Plant Science, Volume 163, Issue 6, 2002. - Iwao, Kyoko, Watanabe, Takashi, Fujiwara, Yoshiyuki, Takami, Koji, Kodama, Ken, Higashiyama, Masahiko, Yokouchi, Hideki, Ozaki, Kouichi, Monden, Morito, Tanigami, Akira. Isolation of a novel human lung-specific gene, LUNX, a potential molecular marker for detection of micrometastasis in non-small-cell lung cancer. International Journal of Cancer. Int. J. Cancer) Comment No.2: Based on figure 2, all lanes 2, 3 and 4 showed the smear, and, intriguingly, only the 150 uM resulted in the amplification of the TDAT gene. Response: As you know, the smear is commonly seen in the background of agarose gel electrophoresis of RT-PCR products. Moreover, In the case of this study, it was very difficult to obtain RNA samples with high concentrations of total RNA from cultured cells of hazelnut. For this reason, we had to use maximum amounts of total RNA samples and RT products in RT and subsequent PCR reactions. These conditions were the main reasons for the observation of smear in the background of agarose gel. The authors also, carried out this experiment aimed at significant expressing of the TDAT gene, several times and it was confirmed from the results that the expression was not detected at low concentrations of elicitors. About the amplification of desired PCR products only in reactions with RNA from 150 µM MeJA, Nevertheless, according to the dear reviewer comment, and to avoid of the claims related with comparative experiments, the phrase ‘while TDAT expression was induced at only 150 µM of MeJA’ was revised with the phrase ‘and it was perceivable at higher concentration of MeJA’(P8 line219 and 220), and the phrase “of an exact concentration” was deleted from P19-line 507 Comment No.3: What is the dark line present in figure 2 between lane 3 and lane 4? The authors claimed that gel cropped from different gels was differentiated by white lines; however, no such differentiation exists in this figure. Response: That's correct, and thanks for bringing it out. The trimmed areas of figure 2 had been distinguished in the original figure (S1_ row_ images.pdf) attached to the supplementary information. So the change was considered, and the dark line was replaced with white lines. During the previous edition, the figure 2 was also modified in response to feedback from reviewers. Comment No.4: The authors claimed that they had purified the PCR product obtained from lane 5. Was it gel elution or purification?. Response: It was gel purification using Ron’s Gel Extraction Kit (BIGRON, cat no.: 802501) for 1423 bp. It is mentioned at page 7 line 155. Comment No.5: P7 L155-authors mentioned as gel extraction- these details may be precisely given in the results section.. Response: Many thanks for the comment. The phrase “using Ron’s Gel Extraction Kit (BIGRON, cat no.: 802501)” was deleted from the results. Comment No.6: Moreover, lane 7 does not seem to contain the purified products, instead, it looks more smear than the unpurified products (Lane 5). The authors explain this context. Response: Many thanks for these precise comments. As previously stated, the 1423 bp amplified DNA fragment was purified from an agarose gel using Ron’s Gel Extraction Kit (BIGRON, cat no.: 802501). Despite all the optimizations, the target PCR fragment was so thin that we had to use a large gel slice in the purification process. The smear in the background of Lane 7 is caused by utilizing the maximum amount of an agarose gel in the purification process. It's also worth noting that we designed PCR primers using the TDAT assembly sequence derived from an EST library as a template. To amplify the complete CDS in PCR reactions, we had to locate the primers at the end of the target sequence. As a result, we faced significant challenges in detecting and overcoming false priming and other qualitative criteria in designed primers. Undesirable bands have been amplified in PCR reaction possibly due to the false priming of PCR primers. However, we obtained a clean PCR product when using the purified PCR product as the template in nested-PCR reactions (S3 Fig, page 9). Comment No.7: Based on figure 2, the presence of TDAT amplicon in the lanes 2, 3 an 4 can not be ruled out. There might be some problem with the purification of PCR amplicons from the gel. Collectively, to avoid all these misperceptions, the authors are highly recommended to perform real-time quantitative PCR analysis to detect the relative mRNA level of TDAT gene at 0, 50, 100 and 150 uM of MeJa. Response: Thank you so much for your detailed comment. Certainly, qRT-PCR is most reliable choice for comparative expression studies of TDAT gene. However, as stated in the title, the major goal of this study was identification and sequence analysis of the target gene in hazelnut. Therefore, we focused our efforts on providing a detectable source of TDAT mRNA for use in gene isolation assays. We performed many PCR reactions on different amounts of RT samples from different cell samples. Every time, we found the target mRNA only in cells induced with 150 µM MeJA. With this reason, the work is only based on common RT-PCR reactions not semi quantitative or quantitative ones. It was enough to our purpose of the study. We checked throughout the manuscript and made essential revisions to ensure there are no claims related with comparative experiments. Please refer to P8 line 219 and 220; P19-line 507 in the revised manuscript. Comment No.8: The number of figures may be reduced. Few figures may be shifted to the Supplementary file. For instance, Figs 1, 2, 3, 5, 7 Response: The modification was considered and the fig 1, 2, 5 and 7 were shifted to the supplementary file. Comment No.9: P4 L96: What is the content/quantity of callus taken to generate suspension culture as per the given protocol, which may be provided in the methods section. Response: Almost 1 g white and friable callus was taken and suspended in 30 mL liquid media to generate suspension culture. The modification was added to the text. Comment No.10: Which term the authors imply: produced or procured? Response: The term produced was implied. It was recommended with the other reviewer. Comment No.11: µm Response: Thanks to the reviewer for the comment. The modifications have been considered. Comment No.12: Primer sequences may be given in the table Response: The primers’ sequence was added in S1 table. Comment No.13: P19 L454-455: This claim warrants the use of qPCR analysis, considering the inconsistent result of the given sqRT-PCR gel pictures. Response: Thanks for these precise comments from the reviewer. The authors did not do quantitative analysis because their efforts were focused on obtaining a reliable and detectable supply of TDAT mRNA for use in gene isolation assays, as stated in earlier comments. Using different concentrations of elicitors, in this paper, indicates valuable results of using elicitation for identification of low-expressed taxol genes in hazelnut or even other genes. Please consider our more explanations under the reviewer suggestions about qPCR. Submitted filename: responses to reviewer.docx Click here for additional data file. 13 Aug 2021 ​Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L. PONE-D-21-05459R2 Dear Dr. Rahpeyma, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Balamurugan Srinivasan Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #3: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: (No Response) ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: (No Response) ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #3: (No Response) ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #3: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #3: Since the authors clarified all the queries raised by this reviewer and also considering the revisions accorded in the submitted version, the manuscript in its present form may be accepted for publication. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #3: No 19 Aug 2021 PONE-D-21-05459R2 Identification and in-silico characterization of taxadien-5α-ol-O-acetyltransferase (TDAT) gene in Corylus avellana L. Dear Dr. Rahpeyma: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Balamurugan Srinivasan Academic Editor PLOS ONE
  36 in total

1.  Taxol biosynthesis and molecular genetics.

Authors:  Rodney Croteau; Raymond E B Ketchum; Robert M Long; Rüdiger Kaspera; Mark R Wildung
Journal:  Phytochem Rev       Date:  2006-02       Impact factor: 5.374

2.  Coevolutionary analysis of resistance-evading peptidomimetic inhibitors of HIV-1 protease.

Authors:  C D Rosin; R K Belew; G M Morris; A J Olson; D S Goodsell
Journal:  Proc Natl Acad Sci U S A       Date:  1999-02-16       Impact factor: 11.205

3.  Transcriptome profiling reveals specific patterns of paclitaxel synthesis in a new Taxus yunnanensis cultivar.

Authors:  Chun-Tao He; Zhi-Liang Li; Qian Zhou; Chuang Shen; Ying-Ying Huang; Samavia Mubeen; Jun-Zhi Yang; Jian-Gang Yuan; Zhong-Yi Yang
Journal:  Plant Physiol Biochem       Date:  2017-11-15       Impact factor: 4.270

4.  Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

Authors:  Q Yang; K Reinhard; E Schiltz; U Matern
Journal:  Plant Mol Biol       Date:  1997-12       Impact factor: 4.076

5.  Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

Authors:  Yechun Wang; Zhiqi Miao; Kexuan Tang
Journal:  Mol Biol Rep       Date:  2009-12-10       Impact factor: 2.316

6.  Taxanes from Shells and Leaves of Corylus avellana.

Authors:  Laura Ottaggio; Federica Bestoso; Andrea Armirotti; Alessandro Balbi; Gianluca Damonte; Mauro Mazzei; Monica Sancandi; Mariangela Miele
Journal:  J Nat Prod       Date:  2007-12-29       Impact factor: 4.050

7.  The final acylation step in taxol biosynthesis: cloning of the taxoid C13-side-chain N-benzoyltransferase from Taxus.

Authors:  Kevin Walker; Robert Long; Rodney Croteau
Journal:  Proc Natl Acad Sci U S A       Date:  2002-06-27       Impact factor: 11.205

8.  Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway).

Authors:  Yechun Wang; Binhui Guo; Fei Zhang; Hongyan Yao; Zhiqi Miao; Kexuan Tang
Journal:  J Biochem Mol Biol       Date:  2007-11-30

9.  In vitro cell cultures obtained from different explants of Corylus avellana produce Taxol and taxanes.

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Journal:  BMC Biotechnol       Date:  2006-12-06       Impact factor: 2.563

10.  Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

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Journal:  Biotechnol Bioeng       Date:  2020-10-07       Impact factor: 4.530
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