Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L.

Accepted Manuscript Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L. Fatemeh Khakdan, Houshang Alizadeh, Mojtaba Ranjbar PII:

S0981-9428(18)30326-7

DOI:

10.1016/j.plaphy.2018.07.026

Reference:

PLAPHY 5349

To appear in:

Plant Physiology and Biochemistry

Received Date: 25 March 2018 Revised Date:

20 July 2018

Accepted Date: 22 July 2018

Please cite this article as: F. Khakdan, H. Alizadeh, M. Ranjbar, Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L., Plant Physiology et Biochemistry (2018), doi: 10.1016/j.plaphy.2018.07.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Molecular cloning, functional characterization and expression of a drought inducible phenylalanine ammonia-lyase gene (ObPAL) from Ocimum basilicum L.

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Fatemeh Khakdan1, Houshang Alizadeh2, Mojtaba Ranjbar3*

1- Biotechnology Department, College of Agriculture, Jahrom University, Jahrom, Iran

2- Division of Molecular Plant Genetics, Department of Agronomy & Plant Breeding, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran

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3- Microbial Biotechnology Department, College of Biotechnology, University of Special

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Modern Technologies, Amol, Iran

* Corresponding author: Mojtaba Ranjbar

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Email address: [email protected]

ACCEPTED MANUSCRIPT Abstract Phenylalanine ammonia-lyase (PAL) is a control point for branched phenylpropanoid and terpenoid pathways. It represents the first regulatory step to provide a metabolic flux to produce of the precursors needed for biosynthesizing main volatile phenylpropanoid compounds (methyleugenol and methylchavicol) in basil. It is crucial during the stage of the

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environmental and development stimulants. To obtain better knowledge of the biosynthesis of these phenylpropene compounds, characterization and cloning of Ocimum basilicum PAL (ObPAL) cDNA and its heterologous expression and enzyme activity were assessed. The almost full-length ObPAL was 2064 bp in size encoding a 687-amino-acid polypeptide with

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molecular weight of 74.642 kDa and theoretical pI of 8.62. Phylogenetic analysis revealed a significant evolutionary relatedness of ObPAL with the PAL sequence reported in different species of Lamiaceae. To further confirm its function, ObPAL was cloned into pET28a (+)

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vector and expressed in E. coli. The recombinant protein exhibited high PAL activity and could catalyze the L-Phe conversion to trans-cinnamic acid. Expression analysis of PAL gene showed that ObPAL manifested various transcription ratios exposed to drought stress. Overall, our results demonstrated the ObPAL regulation gene is possibly a mechanism dependent on cultivar and drought stress.

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expression, drought stress

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Keywords: Ocimum basilicum L., Phenylalanine ammonia-lyase, Cloning, Characterization,

ACCEPTED MANUSCRIPT Abbreviations Analysis of variance

COMT

Caffeoyl-CoA O-methyltransferase

C4H

Cinnamate 4-hydroxylase

C3H

p-coumarate 3-hydroxylase

4CL

4-coumarate-CoA ligase

CVOMT

Chavicol O-methyltransferase

HAL

histidine ammonia-lyase

LB

Luria-Bertani

IPTG

Isopropyl-β-D-thiogalactoside

NLS

Nuclear Localization Signals

PAL

Phenylalanine ammonia-lyase

Phe

Phenylalanine

RACE

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Subcellular location prediction

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PLS

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ANOVA

Rapid amplification cDNA ends

ACCEPTED MANUSCRIPT 1. Introduction Basil (Ocimum basilicum L., Lamiaceae family) is a well-known herbaceous, aromatic and medicinal plant. Since more than 2,000 years it has been used in Ayurvedic, other traditional

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systems of medicine for the treatment of nervous and digestive disorders (Moghaddam et al. 2014). According to the pharmacological surveying, the wide array of the main bioactive volatile compounds in the O. basilicum essential oil are divided into two main groups: one is terpenoids group, and the other phenylpropanoids group that are synthesized and stored in

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specialized glands known as glandular trichomes on the surface of their leaves, which all

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critically play in the particular properties of many spices and herbs (Xie et al. 2008). Methyleugenol and the related phenylpropense, methylchavicol are the most important flavor compounds in certain varieties of O. basilicum, which have the central role in their notable pharmacological, structural properties and defense functions (Gang et al. 2002; Šimović et al. 2014).

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The extensive utility of culinary and medicinal properties of O. basilicum in traditional systems of medicine and recent enthusiasm regarding the crucial role of the aromatic and

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bioactive properties as pharmaceutical and defensive materials in basil has made researchers illustrate the methyleugenol and methylchavicol pathway. As the detailed the preferred

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mechanisms of their biosynthesis regulation should be completely comprehended, it is all the more imperative to identity, characterize, and clone major genes of pathway contributing to the production, modification and developmental regulation of phenylpropanoid pathway metabolites at molecular levels to ascertain many enzymes’ controlling role in methyleugenol and methylchavicol biosynthesis. In plants, the biosynthetic pathway of volatile phenylpropenes i.e., methyleugenol and methylchavicol are still unclear, and they are shared with monolignols’ formation required for lignin/lignan biosynthesis in the preliminary steps (Koeduka et al. 2006) wherein,

ACCEPTED MANUSCRIPT phenylalanine ammonia-lyase (PAL) is the first, branching point, important regulatory enzyme of phenylpropanoid pathway in regulating over flux the carbon flux into subsequent biosynthetic of various volatile phenylpropane compounds pathways (Campos et al. 2004; Vogt, 2010) by de-amination, addition of hydroxyl and methoxyl functionalitie groups,

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methylation and decarboxylation steps (Vogot, 2010). As the entry point into the pathway, PAL catalyzes L-phenylalanine deamination to produce ammonia and trans-cinnamic acid, a substrate common to the biosynthesis of different classes of phenylpropanoid products e.g.

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methyleugenol and methylchavicol (Gang et al. 2001). The position of PAL at the regulation point connecting general phenylpropanoid metabolism with different of specific products

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pathways also makes this enzyme as a rate-limiting step for intervention, with reference to its crucial performance in the biosynthesis of abiotic environmental-triggered phenolic derivatives (Ritter and Schulz 2004). A perusal of literature reveals, the activity of PAL is highly controlled at the level of transcription and varies greatly depending on the stage of

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development, cell and tissue differentiation associated with cell-type specific synthesis of phenylpropanoid compounds owning to the changes in PAL activity with reference to its significance in the biosynthesis of different secondary metabolites to aid the plant

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development, structural support, and defense responses and endurance against abiotic

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stimulants (Dixon et al. 2002), investigation of structural and regulatory properties of PAL encoding genes have taken place in several higher plant species (Song and Wang 2009; Jin et al. 2013; Zhang et al. 2014; Dehghan et al. 2014). The PAL genes are encoded by small multi-gene family with two to four copies in the haploid genome. Tomato (Solanum lycopersicum) with more than 26 copies and potato (Solanum tuberosum, Solanaceae) with approximately 40–50 copies (Joos and Hahlbrock 1992) are the exceptions (Lee et al. 1992). Key metabolic genes expression in genetic manipulated-prokaryotic expression systems as host plant provides large-scale production of desired therapeutic protein or investigation on

ACCEPTED MANUSCRIPT functional properties of the cloned protein (Yesilirmak and Sayers 2009). As a key enzyme in regulating phenylpropanoid biosynthesis, PAL encoding genes have been cloned and characterized in some cyanobacteria and fungi (Moffitt et al. 2007). Plant PAL gene has also been previously isolated and characterized in Salvia miltiorrhiza (Song and Wang, 2009),

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Mellisa officinalis (Weitzel and Petersen 2010), Angelica gigas (Park et al. 2010), Pinus taeda (Bagal et al. 2012), Juglans regia (Xu et al. 2012), Dendrobium candidum (Jin et al. 2013), Prunella vulgaris (Kim et al. 2014), Solenostemon scutellarioides (Zhu et al. 2015).

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Despite a number of investigations have been previously accomplished for the suitability of PAL gene for pathway engineering in different research areas (Bauer et al. 2011), PAL has

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proved to play an imperative role in production of volatile phenylpropanoid in plants, according to the best our knowledge, though the detailed information and the function of the regulatory role of PAL may to have a cascading influence on the up regulation of downstream genes in the increasing the levels of methyleugenol and methylchavicol as the

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overall metabolite, has not been described in basil. With this in mind, the current evaluation was accordingly aimed at the molecular studies on the PAL from O. basilicum with regard to cDNA cloning, functional characterization as heterologous expression of in vitro enzyme

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reaction and biochemical characterization of the ObPAL gene to gain insights into the

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molecular regulatory mechanisms.

Here, the expression pattern of PAL and accumulation ratio of the methyleugenol and methylchavicol in three basil Iranian cultivars in response to the three levels of drought stress (i.e., mild, moderate and severe) at flowering stage also investigated. As phenylpropanoid pathway generates numerous volatile compounds, evaluates the properties and expression patterns of genes contributing to generation of these metabolites, such as PAL, to realize defense mechanisms against different types of stresses, and the biosynthesis of the compounds is beneficial.

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Plant materials and growth conditions Basil seeds (Ocimum basilicum L.), from two regions of Iran [i.e., Amol (Cultivar 2 and 3) and Jahrom (Cultivar 1) cities], were sown in individual pots filled with sandy-loam soil

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located at the research greenhouse of Faculty of Agriculture, University of Tehran, Karaj, Iran (35°49′57″ N, 50°59′29″ E), with the beginning time of Mid-May, 2016. The two-weekold seedlings were used for extraction of RNA and various drought treatments. Three

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different stress levels were adjusted first in relation to field capacity (FC) via withholding water, described as C: 100% FC, W1: 75% FC, W2: 50% FC and W3: 25% FC (Khakdan et

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al. 2016; Khakdan et al. 2017). After conducting 29 days drought stress treatment, three cultivars were sampled at complete blooming phase (May to June, 2014). The shoot specimens of every treatment were collected and immediately frozen in liquid nitrogen and

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stored at -80 °C until further analyses.

2.2. Isolation of DNA, RNA and cDNA synthesis Total isolation of RNA was done by a pBiozol (Sigma) reagent based on the protocol

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according to manufacturer's instructions. cDNA was synthesized with the Revert-AidTM

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First Strand cDNA Synthesis Kit (Fermentas GmbH, St. Leon-Rot, Germany) based on the manufacturer's recommendations. Total DNA was separated from leaves of the cultivars essentially by a modified procedure (Haque et al. 2008).

2.3. Cloning of a PAL cDNA A pair of degenerate oligonucleotide primers (ObPALf, ObPALr; see Table 1) designated based on the conserved sequence motifs of reported several plants were used for the

ACCEPTED MANUSCRIPT amplification of the core cDNA fragment of ObPAL. PCR was used with the following program applying cDNA as a template: 32 cycles of 94 °C for 2 min, 53 °C for 1 min, and 72 °C for 1.30 min followed by a final extension of 72 °C for 10 min. An amplicon of 574 bp was recovered from the agarose gel, ligated into pTG19-T PCR cloning vector (Vivantis,

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Korea), transformed in DH5α cells (Novagen, USA) and the isolated plasmid was employed for determination of sequence (Bioneer Biotechnology Co. Ltd (Korea)). The PCR product for partial ObPAL designed the nested gene-specific primers [forward (Ob-PAL1, Ob-PAL2)

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and reverse (R1) primers] for the cloning of 3' end of ObPAL by 3' RACE-PCR with the conditions as follows: 30 cycles of 94 °C for 2 min, 59 °C for 1 min and 72 °C for 1.30 min

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followed by an ultimate extension for 5 min at 72 °C. The amplified fragment was eluted from the gel using glass-milk method and cloned into pTG19-T PCR cloning vector, and the subjected to nucleotide sequencing. The obtained nucleotide sequence for partial ObPAL showed it shared maximum identity (> 80% identities) with the PAL sequence reported in

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Perilla frutescens (data are not given). So for full-length sequence, ObPAL-P1 and ObPALP2 primers (as the initial primer of amplification and nested primer, respectively; Table 1) as forward primer were directly synthesized from Perilla frutescens PAL sequence. The reverse

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primer was synthesized according to the data from previously cloned fragments (Table 1).

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Reactions of touchdown-PCR were done for 1 min at 94 °C, followed by 94 °C for 30 s; 62 °C for 1 min, and 72 °C for 1 min in the initial cycle. The anneal temperature declined 1 °C in every cycle. The conditions were altered to 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min for 24 cycles, and then a final extension at 72 °C for 7 min after 6 cycles. The nested amplified parts were purified, ligated, cloned and sequenced as above-mentioned.

ACCEPTED MANUSCRIPT 2.4. Sequence analysis The near full-length of cDNAs of the ObPAL gene were gained by SeqMan and EditSeq software to align the cloned gene components, compared based on web database via the program of BLAST accessible on the website of NCBI (http://www.ncbi.nlm.nih.gov/).

found

using

corresponding

bioinformatics

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Searches for the longest open reading frame (ORF) in all six possible reading frames was software

(http://www.

Ncbi.nlm.nih.gov/gorf/gorf.html). The prediction of in silico translation of the sequences of into

sequences

of

amino

acid

was

done

by

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nucleotide

translate

tool

(http://www.expasy.ch/tools/dna.html) and the physiochemical features of ObPAL amino sequence

were

analyzed

using

ScanSite

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acid

and

ProtParam

(http://www.Expasy.ch/tools/protparam.html) to obtain pI, molecular weight, etc. The structural and functional important regions (motif prediction), conserved domains, protein domain families, catalytic domains were detected in the protein sequence via PROSITE,

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InterProScan, ProDom, TrEMBL, respectively. For thorough analyzing of the conserved domains, MotifScan was used to identity catalytic domains and analyze any motif rearrangement, the methyltransferase catalytic domain class, ATP-GTP binding motif, and

program

SOPMA

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modeling

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metal binding domain. Also, secondary structure was indicated using the protein structure

hydrophobicity

analysis

(http://npsa-pbi.ibcp.fr).

was

done

by

using

Locality, Antheprot

signal

survey

software;

and

SignalP

(http://www.cbs.dtu.dk/services/SignalP/), PSORT Prediction (http://psort.hgc.jp/form.html), predict Nuclear Localization Signals (NLS) tool (http://predictprotein.org/) and Subcellular location prediction (PLS) web tools.

ACCEPTED MANUSCRIPT 2.5. Phylogenetic analysis and prediction of the three-dimensional structure of ObPAL The ObPAL three-dimensional structure was estimated by MODELLER based on the wide range of similar sequences of PAL from different organisms. The program PROCHECK was used to the check and validate the possible designed three-dimensional structure and specified

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pattern in the protein sequence.

The ObPAL amino acid sequence and other PAL genes with the greatest grade from several plants from the GeneBank through the BLASTp were aligned with CLUSTAL W tool. A

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MEGA 6.0 program and by neighbor-joining method.

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phylogenetic tree was made based on analysis of bootstrap with 100 replicates with the

2.6. Heterologous expression of ObPAL in Escherichia coli BL21 (DE3) The partial of open reading frame (ORF) was amplified with primers ObPAL-SalI–F and ObPAL-NotI-R, employing the first strand cDNA as template (Table 1), that contained,

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respectively, SalI and NotI restriction sites (underlined). The resulting amplicon with double restriction site was first ligated into sequencing vector of pTG19, digested through appropriate sites. The resultant digested yield was cloned in pET2-28a(+) expression vector

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(Novagene, USA) with the mentioned restriction enzymes. The resulting recombinant

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plasmid, pET2-28a (+)-ObPAL, was then sequenced to confirm for the correction of the ORF. Subsequently, the expression constructs pET2-28a(+)-ObPAL was put on E.coli BL-21 (DE3) cells for protein expression. A single colony of E.coli BL-21 (DE3) cells with the expression plasmid pET2-28a(+)-ObPAL was cultured at 37 °C in fresh Luria-Bertani (LB) medium containing kanamycin (100 mg/L), with shaking (180 rpm) till the OD600 reaches about 0.6. Then, the expression of protein was triggered via addition of isopropyl-β-Dthiogalactoside (IPTG, Merk, Germany) to an ultimate 1 mM concentration and kept at 30 °C for 4, 5 and 7 h. The resulting recombinant cells were grown via centrifugation at 4 °C for 20

ACCEPTED MANUSCRIPT min at 4000 g. The pellet was thawed and suspended in 1 mL lysis buffer (PBS; 50 mM, pH 7.4), and then sonication was applied to disrupt the cells (3 × 30 s) at 4 °C. The sonicated sample was centrifuged at 12000 g for 10 min at 4 °C. The protein expression level was measured by Bradford method (Biorad, USA) and analyzed on 10% SDS-PAGE by

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Coomassie Blue staining, then the clear protein extract subjected to enzyme activity assay or frozen and kept at -20 °C to be later used.

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2.7. Enzyme activity assays

The resulting protein extract of recombinant construct (pET2-28a(+)-ObPAL) examined the

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PAL activity using (Song and Wang 2009) method with minor modifications. The reaction assay of the PAL assay consisted of 2 mL 0.01 M borate buffer (pH 8.7), 50 µL crude protein extract, and 1 mL 0.02 M L-Phenylalanine (pre-dissolved in 0.01 M borate buffer of pH 8.7), while controls without the substrate was performed. For optimum temperature estimation, the

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reaction was incubated at a constant pH of 8.7, while varying temperatures (50, 60, and 70 °C) for 30 min and then the assay was stopped by incubating on ice. The ObPAL activity was

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290 nm.

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detected spectrophotometrically through measurement of the reaction mixture absorbance at

2.8. Expression analysis using quantitative real time PCR (qRT-PCR) Total RNA was obtained from young leaves of the cultivars of basil at the three levels of drought stress to study the expression profiles of ObPAL, and 1.0 µg of this RNA was transcribed into cDNA as mentioned above. The primer sequence of every primer was designed

based

on

the

corresponding

gene

using

Primer3

software

(http://frodo.wi.mit.edu/primer3/) (Table 1). Conventional PCR was employed first using a Bio-Rad thermocycler in 25 µL containing 1X PCR buffer, 0.4 µM of every primer, 0.2 mM

ACCEPTED MANUSCRIPT of dNTPs, 200 ng of cDNA and 1 unit of Taq DNA polymerase (5U/µL). A temperature profile of 5 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at the specific annealing temperature for each primer, 60 s at 72 °C, and a final extension for 10 min at 72 °C. The amplicons, in the following, were electrophoresed on 2% agarose gel and observed

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through an ethidium bromide based staining system. Next, qRT-PCR amplification assay was performed via a QIAGEN's real-time PCR system (Rotor-Gene Q), in an ultimate 20 µl reaction volume containing 8 µL EvaGreen Master Mix (containing EvaGreen Dye, Solis

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BioDyn, Germany), 1.5 µL of diluted cDNA, 0.25 µM of each primer followed by adding PCR-grade water. The qPCR was done with a temperature profile of 15 min at 95 °C, 45

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cycles of 15 s at 96 °C, 20 s at the specific annealing temperature for each primer, and 20 s at 72 °C. Additionally, the Actin gene (Genbank Accession No. AB002819) was also used as housekeeping gene (Table 1) (Renu et al. 2014) and the expression quantity of the samples obtained in control statuses were set as the reference value. The efficiency of PCR was

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obtained by QIAGEN’s real-time PCR machine (Rotor-Gene Q) for every reaction; values identical to or greater than 0.8 (80%) were used for extensive analyses (Khakdan et al. 2017). The experiments were repeated at least thrice, and statistical analysis of gene expression was

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done by REST software (http://rest.gene-quantification.info).

2.9. Statistical analysis

Experiment results were the mean of three replicated treatments. The data were subjected to analyze using analysis of variance (ANOVA) to assess the significant differences between treatments using Duncan's multiple range tests (p≤ 0.05). Also, the values are expressed as the mean ± standard deviation (SD)

ACCEPTED MANUSCRIPT 3. Results 3.1. Cloning of the near full-length ObPAL Degenerate oligonucleotide PCR primers constructed from an initial core fragment known consensus sequence amplified (~ 574 bp) of a putative PAL cDNA from O. basilicum leaf

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which blast results showed maximum identity with Perilla frutescens. RACE-PCR estimated the 3' ends of core fragment. For near full length PAL, PCR was done with various sets of gene primers designed from partial length sequence of core amplicon and 5' end sequence of

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Perilla frutescens PAL gene. It is designated as ObPAL and registered in GeneBank (Accession No. KU375119). The near full-length cDNA of ObPAL was 2229 bp in size

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encoding a protein of 687 deduced amino acids having an ORF of 2064 bp, flanked by 165 bp of a 3' UTR followed by 133 bp polyA tail.

3.2. Analysis of the deduced protein sequence

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Based upon sequence homology tools (Blast P online (http://www.ncbi.nlm.nih.gov/BLAST) and multiple alignment analysis by Clustal W, the ObPAL amino acid sequence revealed highly identity with PALs reported in plants in the GeneBank database, sharing 86% identity

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to PfPAL (JQ277717.1), 85% identity to MoPAL (FN665700.1), 83% identity to ArPAL

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(AF326116.1), 82% identity to SmPAL (DQ408636.1), 81% identity to SbPAL (HM062777.1) (Fig. 1). A phylogenetic tree was made on the basis of the ObPAL and known sequences of PAL from various different plant species by using MEGA 6.0 tool (Fig. 2). The phylogenetic result indicated different PAL forms evolving from a single ancestral gene and the ObPAL was a PAL family member, suggesting that the degree of relatedness correlated well with the amino acid similarity among the members of PAL family. Phylogenetic tree was classified into four clusters. All of the clusters were composed of dicots. ObPAL falls into the near cluster 4 and was found associated with P. frutescens followed by M.

ACCEPTED MANUSCRIPT officinllalis (Fig. 2). The ObPAL protein molecular mass was predicted to be about 74.64 kDa and the isoelectric point was predicted to be at 8.64 calculated by the Online Computer pI/MW Tool (http://cn.expasy.org/tools). In addition, the putative amino acids sequence revealed more homology with conserved active site consensus sequence of all the PAL conserved

motifs

([STG]-[LIVM]-[STG]-[AC]-S-G-[DH]-L-X-P-L-[SA]-X-X-

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protein

[SAV]) was also detected in the ObPAL protein (Fig. 1) (Mahesh et al., 2006). This broad similarity was seen for 25-523 residues; this stringent conservation in the PAL family might

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show the functional importance of these amino acids, whether can be as a protein marker for PAL enzyme. Also, the multi-alignment results revealed that amino acids at the N-terminal

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area of the protein showed least sequence identity in length and the composition. ObPAL shows specific hits with two major motifs, histidine ammonia-lyase, and belongs to the lyaesI-like superfamily (consisting of 512 amino acids, from position 1 to 512) incorporating PAL, HAL (histidine ammonia-lyase), and phenylalanine and histidine ammonia-lyases signature

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(consisting 16 amino acids, from position 168 to 184). According to conserved domain database, the ProDom and TrEMBL results showed ObPAL protein consist of the essential domain; the de-amination conserved (L203, V169, A248), conserved active site catalytic

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(N284, G146, G253, Q342, Y218 and DND [389-391]) and five highly conserved peptide

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domains (HNQDV [493-497]) have been specified in the known phenylalanine ammonialyase (Fig. 1). These domains are important as substrate binding pocket, catalytic active site, effective agent in 4- methylidene imidazole-5-one (MIO) forming and chemical binding site which consists of about 13 amino acids distributed throughout the polypeptide. The amino acid residues in these domains are also conserved in the PAL family polypeptide. MotifScan results revealed that the protein sequence of ObPAL had 11 catalytic domains consisting of eight different catalytic domains, viz. ASN_GLYCOSYLATION (N-glycosylation site), CAMP_PHOSPHO_SITE (cAMP- and cGMP-dependent protein kinase phosphorylation

ACCEPTED MANUSCRIPT site), CK2_PHOSPHO_SITE (Casein kinase II phosphorylation site), MYRISTYL (Nmyristoylation site), PKC_PHOSPHO_SITE (Protein kinase C phosphorylation site), TYR_PHOSPHO_SITE (Tyrosine kinase phosphorylation site), HUTH_MF_00229 Histidine ammonia-lyase [hutH],

PAL_HISTIDASE Phenylalanine and histidine ammonia-lyases

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signature. Using PROSITE motif search (http://www.expasy.ch/tools/) and InterProScan tool (http://www.ebi.ac.uk/interpro), ObPAL protein sequence predicted several consensus protein motifs, which included conserved motif GTITASGDLVPLSYIAG (206-221 aa) contained an

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Ala-Ser-Gly tripeptide (209-211), the highly conserved residues. Furthermore, the secondary structure analysis of ObPAL was done by Self-Optimized Prediction Method with Alignment

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(SOPMA) (http://expasy.org/tools/SOPMA). PSORT analysis showed the presence of a dileucine motif LL (222-223) in the N-terminal region of ObPAL amino acid sequence, which was not found in the protein localization signal peptide at the C-terminal region. Results revealed that predicted ObPAL protein was a predominantly α-helical protein, which mainly

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consisted of alpha helices (52.26%) and random coils (27.22%), extended strands (10.63%), and 9.90% beta turns. In order to better characterize the ObPAL protein, the ObPAL threedimensional design generated by MODELLER, while the validity of the 3D construct was

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confirmed by PROCHECK. The 3D structures of ObPAL were shown through UCSF

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Chimera (https://www.cgl.ucsf.edu/chimera) in Fig. 3. The location establishment of the predicted protein was checked by NLS and PLS analysis tools. Locality, signal survey and hydrophobicity analysis revealed at least one cytosolic helices signal peptide towards the Nterminal of the sequence and possibility anchoring protein to endoplasmic reticulum.

3.3. Bacterial expression and characterization of ObPAL To examine the ObPAL expression in E.coli, the ORF sequence of ObPAL was inserted into expression vector pET28a(+), the pET- ObPAL structure was sequenced to examine in-frame

ACCEPTED MANUSCRIPT fusion and changed into E.coli BL-21 (DE3) cells. SDS-PAGE analysis, visualized by Coomassie Brilliant Blue R250 staining, revealed that of the resultant ObPAL fusion expressed a recombinant protein whose molecular mass was an approximately 74.642 kDa (Fig. 4). It was in good agreement with that predicted by bioinformatics method. Moreover,

conversion

of

L-Phenylalanine

to

trans-cinnamic

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to confirm ObPAL as a functional gene and whether the protein are active or not, the acid

was

also

measured

spectrophotometrically at 290 nm. The different time course for expression was also

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examined. The data revealed that the maximal level of the protein expression was achieved at 7 h after IPTG induction (data not shown). The highest activity of the ObPAL was shown in

3.4. Transcription profile of ObPAL

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borate buffer at the optimal pH 8.7 and the optimal temperature was at only 60 °C (Fig. 5).

Relative expression analysis of ObPAL transcripts accumulation in drought stress was

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analyzed by real-time qRT-PCR. The actin gene transcript level was set as the control to normalize the level of expression remained stable throughout under different levels of drought stress. As Fig. 6 is shown, for both Cul. 1 and 3, relative expression ratio of PAL

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elevated from the beginning of drought stress (W1; 1.25-and 1.39- fold, respectively), and

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gradually increased for W2 (2.38- and 2.98- fold, respectively) and continued ascendingly until occurring severe stress level (W3; 3.78- and 4.012-fold, respectively), while regarding the second cultivar, Cul. 2, expression ratio of PAL was gradually up-regulated during the first (W1; up to 2.12-fold change) and second (W2; up to 2.48-fold change) levels of drought stress treatment and increased sharply upon the third of drought treatment with the fold change value of ~6.27.

ACCEPTED MANUSCRIPT 4. Discussion Although O. basilicum contains a number of secondary metabolites, namely terpenoids, phenylpropanoids and phenolic compounds, carotenoids and vitamins, methyleugenol and methylchavicol as phenylpropanoid-derived natural compounds have often been observed as

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the main constitutes of basil essential oil (Pirbalouti et al. 2013). Though the plant, O. basilicum has been well characterized in terms of pharmaceutical activities (Makari and Kintzios, 2008), phytochemical profiles (Hassanpouraghdam et al. 2010; Pirmoradi 2013) as

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well as gene expression profiles (Renu et al. 2014; Rastogi et al. 2014), little is known regarding the general key genes responsible for biosynthesis of these compounds. Researches

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have revealed that PAL is as an enzyme of the biosynthesis in the main fragrances phenylpropanoids compounds in glandular peltate trichomes of Ocimum basilicum (Xie et al. 2008; Rastogi et al. 2014). Since then, to date, more PAL gene have been indicated and cloned from several different species. But in O. basilicum, a significant aromatic plant, there

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is little detailed data describing PAL gene so far. To gain new insights regarding the impact of PAL on the pathway of phenylpropanoid biosynthesis in O. basilicum, the full length, or near full-length gene cDNA sequence of ObPAL gene was cloned and specified. The in silico

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analysis of the O. basilicum PAL sequence showed that the obtained sequence sharing more

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identities/ resemblance to sequences of PAL from other species. The PAL amino acid sequence from Perilla frutescens (AEZ67457), Salvia miltiorhiza (ABD73282), Melissa officinalis (CBJ23826) is 89% identical with the sequence from O. basilicum and 88%, 86% to the putative PAL sequence from Sesamum indicum (XP011094662) and Erythranthe guttata (EYU38541). ObPAL gene is registered in the GeneBank and characterized bioinformatically, with emphasis on the functional and structural analysis of the gene. The predicted PAL protein had HAL and PAL domains of which the major was Lyase class I domain such as superfamily that is present in plants, fungi, several bacteria and animals.

ACCEPTED MANUSCRIPT PALs, active like homotetramers, catalyze the L-Phenylalanine conversion to trans-cinnamic acid. This enzyme, like other homotetrameric enzymes in this superfamily possess the enzyme's active site that was associated with a serine residue that is converted autocatalytically in MIO group formation with internal cyclization and dehydration of an

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electrophilic tripeptide having serine, amino acids alanine, and glycine (Ritter and Schulz, 2004). This tripeptide is also part of the domain conserved in the ObPAL (Fig. 1a), which are responsible in catalysis and functioned as a pivotal cofactor of PAL in metabolism and

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transport of amino acid in plant (Ritter and Schulz 2004; MacDonald and Dcunha, 2007). The crystal structure of PALs (Fig. 3), which assumed a sea-horse shape (MacDonald and

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Dcunha, 2007) have been recently described in Salvia miltiorrhiza (Song and Wang, 2009), Mellisa officinalis (Weitzel and Petersen, 2010), Juglans regia (Xu et al. 2012); Solenostemon scutellarioides (Zhu et al. 2015). Sequence alignment analysis of PAL amino acid sequences reveals the most of catalytic active site amino-acid residues are also

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detectable in the sequence of ObPAL (Fig. 1a) that has a similar function to that of the other PAL, which are assumed to play important roles in the function of this protein (Song and Wang, 2009; Jin et al. 2013; Dehghan et al. 2014) and as were additional residues previously

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noted as conserved in PAL proteins in various plants. Conserved domain database search

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showed several conserved peptide domains in ObPAL in line with PAL from Salvia miltiorrhiza, Mellisa officinalis, Juglans regia, Ginkgo biloba, although there are differences in some residues of amino acid from these domains. Jin et al. (2013) study showed cloned the gene of PAL from Dendrobium and suggested that the enzyme consisted deamination and the catalytic sites to affect this protein’s function. Comparison of the PAL protein sequences of cloned genes has showed the presence of conserved peptide motifs in the C-terminal region of the amino acid of PAL of probable functional significance which was conserved highly in plant PALs as confirmed by the phylogenetic analysis (Song and Wang, 2009). As

ACCEPTED MANUSCRIPT demonstrated in the sequence alignment of PAL from different species, ObPAL is composed of the conserved active site motif (206~221, GTITASGDLVPLSYIAG) of the PAL protein, which is a typical protein tag of phenylalanine/histidine ammonia-lyase (Schuster and Rétey, 1994). In general, the topology of the phylogenetic tree resembles to the Lamiaceae branch in

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the dicotyledons group obtained by Wu et al. (2014) and Zhu et al. (2015). ObPAL is clustered together with other PAL from Lamiaceae species, including Perilla frutescens, Salvia miltiorrhiza, Agastache rugosa, Prunella vulgaris, Mellisa officinalis (Weitzel and

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Petersen 2010; Kim et al. 2014). From homology point of view, in subgroup, ObPAL is associated with plant PAL protein (more than 80%) from Lamiaceae family and retains

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considerable identities to M. officinalis and Salvia miltiorrhiza, a known functional PAL gene (Fig. 2). These results indicate that ObPAL had in common evolutionary ancestor with a typical PAL gene derived from Lamiaceae species on the basis of conserved structure and sequence features like conserved motif and amino acid homologies and may have a similar

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function as PfPAL and MoPAL in the phenylpropanoid pathway. According to previous studies around 16 amino acid residues account for the binding of the specific substrate and catalytic activity of PAL enzyme (Ritter and Schulz 2004; Weitzel and Petersen, 2010; Jin et

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al. 2013; Zhu et al. 2015) (displayed in a box in Fig. 1). Up to now, many PAL genes of plant

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have been cloned, characterized and successfully expressed in bacterial hosts, including PALs in Mellisa officinalis, Solenostemon scutellarioides, and Arabidopsis thaliana. To further confirm its function, ObPAL was expressed in E. coli BL-21. The recombinant protein was able to elimination of ammonia from L-Phenylalanine to convert it to a very low extent trans-cinnamic acid. As expected, L-Phenylalanine is accepted as substrate, which obtained ObPAL encoded a functional protein which had high PAL activity. Moreover, the character of ObPAL was analyzed. The optimum recombinant ObPAL activity temperature was 60 °C (Fig. 5) is the same as for SmPAL1 and JcPAL1, slightly higher than the PALs in

ACCEPTED MANUSCRIPT A. thaliana (31-45 °C), M. officinalis and B. oldhamii (50 °C), but close to that reported in P. crispum (58 °C) and Z. mays (55-60 °C). The optima pH of the recombinant protein ObPAL was 8.2, within the range known for plant PALs (Song and Wang, 2009; Zhu et al. 2015). In most species studied so far, PAL is encoded through a small family of two to four members'

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genes. Exceptions are Solanum tuberosum with 40-50 copies (Joos and Hahlbrock, 1992) and Solanum lycopersicum with about 26 copies in haploid genome (Lee et al. 1992). At least three gene copies can be tentatively predicted in the O. basilicum genome, as indicated by

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southern-blot analysis (not shown). To our knowledge, thorough investigations of involvement of volatile phenlypropanoid pathway e.g. methyleugenol and methylchavicol

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biosynthesis in plant responses to drought stress on enzyme and on gene level are not known for O. basilicum to date. In order to acquire an initial insight and specify the PAL involvement in the regulation and response to drought stress in basil, its expression pattern was checked in the three levels. Three Iranian cultivars of O. basilicum were employed as the

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sources of terpenoid and phenylpropanoid production (Khakdan et al. 2016). Consequently, for both cultivar 1 and 3, comparing to the control, the lowest amounts of PAL transcripts were detected for W1 (1.25-and 1.39-fold, respectively), and gradually boosted for W2 (2.38-

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and 2.98-fold, respectively), reaching to its maximum levels for W3 (3.78- and 4.012-fold,

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respectively). The same as cultivar 1 and 3, subsequent to applying drought treatment, the transcript abundance of PAL enhanced (W1; 2.12- fold) and the same trend continued for W2 with the fold change value of 2.48, and lastly reaching to its maximum transcript accumulation for W3 (6.27-fold) (Fig. 6). We hypothesized that the activity of PAL provides a metabolic flux to produce of the precursors needed for biosynthesizing phenylpropane compounds e.g. volatile phenylpropanoids in the essential oil, as is the critical case for other medicinal plants of Lamiaceae family. At the study of Xie et al. (2008), many enzymes including PAL, C4H, 4CL and 1-deoxy-D-xylulose-5-phosphate synthase (DXS)/1-deoxy-D-

ACCEPTED MANUSCRIPT xylulose-5-phosphate reductoisomerase (DXR) were found as the entry and the control points for branched phenylpropanoid and terpenoid pathways, respectively. The high levels of ObPAL in the final level of drought stress (W3) could favor increased needs for methyleugenol biosynthesis that considered as a plant defense mechanism, and the expression

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of PAL gene can be induced by various environmental factors such as light, infection, low temperature and drought stress (Lawton and Lam 1987; Leyva et al. 1995; Hisaminato at al. 2001; Hsieh et al. 2011). In Cul. 1 and 3, ObPAL gene up-regulated under drought-stressed

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conditions, possibly reinforcing the carbon flux towards branches in other phenylpropanoid compounds biosynthesis. The much higher levels (W3; up to 3.78- and 4.012-fold) of the

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transcript abundances in severe level of drought stress compared with the other levels suggesting that eugenol production in basil may modulate through transcriptional regulation of the upstream enzymes involved in the eugenol biosynthesis.

This results is validated by our previous study (Khakdan et al. 2017), which demonstrating a

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positive relationship between the accumulation of methyleugenol and key gene activity e.g. C3H and COMT. For the cultivar 2, expression ratio of ObPAL was gradually up-regulated during the first (W1; up to 2.12-fold change) and second (W2; up to 2.48-fold change) levels

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of drought stress treatment and increased sharply upon the third level of drought stress

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treatment with the fold change value of ~ 6.27. These results indicated as strong possibility of direct/indirect correlation of the ObPAL carbon flux towards methylchavicol biosynthesis. In fact, under W3 treatment, transcript accumulation of the mRNA levels encoding ObPAL gene can enhance at the same time the expression of related genes contributing to methylchavicol production. One of the major phenylpropanoid products of Cul. 2 is methylchavicol and thus its biosynthesis requires programming of gene expression of the critical up-stream genes e.g. Ob4CL and ObCVOMT and subsequently eugenol synthase activities for processes associated with production of volatile compounds that have been validated as plant defense monitored

ACCEPTED MANUSCRIPT changes. The correlation, shown in previous results (Yesilirmak and Sayers 2009), suggests an association among the expression level of ObPAL, 4CL and CVOMT genes as the key factors in the methylchavicol pathway and produced methylchavicol during drought stress (Khakdan et al. 2017). In addition to ObPAL’ role in direct flux into the central volatile

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compound, the observed the high level of ObPAL gene transcripts in Cul. 2 under W3 could reflect a need for lignin production that play various roles in developmental and stress-related processes, have been validated as plant defense monitored changes.

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In conclusion, cloning and characterization of full-length or nearly full-length cDNA sequence of ObPAL gene, and transcriptional expression patterns of PAL involved in the

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biosynthetic pathway of volatile phenylpropanoids were assessed for the first time in three Iranian cultivars of basil under drought stress condition. Our results confirmed that the expression profiles of the genes involved in biosynthesis of volatile compounds is either down- or up-regulated under drought stress condition. Studies on PAL in basil will not only

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facilitate further better compression of this pathway, but also can be served as a steppingstone for making improvements in pharmaceutical and biotechnological application of this

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valuable medicinal plant in the future.

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Compliance with ethical standards Conflicts of interest

The authors declare no competing financial interests. Author contributions

F.K. and M.R. conceived the experiments; H.A. supervised the experiments; M.R. and F.K. analyzed the results; F.K. wrote the manuscript.

ACCEPTED MANUSCRIPT Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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ACCEPTED MANUSCRIPT Table 1. Sequences of primers used in the present study

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Primer sequences 5'-AAG CCG GTG GTG AAG CTC GG- 3' 5'-TGA CGC CGG CRA GCT TGA AGG-3' 5'-AAC ATC ACT CCA TGC CTG C- 3' 5'-GGG AAG CCS GAG TTC AC-3' 5´- ACGCTACGTAAGGCATGACA-3´ 5'-AAG CCG GTG GTG AAG CTC- 3' 5'-GTC AAA TAC GCA ATT CAC TAG CC-3' 5'- ATA GTC GAC ATG GAT CCC TTG AAC TGG GTA ATG-3' 5'-AAG CGG CCG CTT AGC AAA TAG GAA GAG GTG C-3' 5'-CGC CCT TGT CAA CGG AAC TG- 3' 5'-AAG TTG CCA CCG TGC AGA GCC-3' 5'- GGC ACA TCC ATC TTC CAC AG-3' 5'- CTG GTT CCG AGC TCC TCT-3' 5'- TCTATAACGAGCTTCGTGTTG-3' 3'- GAGGTGCTTCAGTTAGGAGGAC-3'

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Primer name ObPALf ObPALr Ob-PAL1 Ob-PAL2 R1 ObPAL-P1 ObPAL-P2 ObPAL-SalI–F ObPAL-NotI-R Ob-P-Pal-F Ob-P-Pal-R Ob-PAL-E1 Ob-PAL-E2 Actin-f Actin-r

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Fig. 1. Multiple clustal alignment of the predicted amino acid sequences of PAL protein sequence of the O. basilicum with the corresponding sequence of other plant species. O. basilicum (GenBank Acc. No. KU375119), A. rugosa (GenBank Acc. No. AF326116_1), R.

ACCEPTED MANUSCRIPT glutinosa (GenBank Acc. No. AF401636_1), S. miltiorrhiza (GenBank Acc. No. ABD73282.1), S. viscidula (GenBank Acc. No. ACR56688.1), S. baicalensis (GenBank Acc. No. ADN32767.1), P. frutescens (GenBank Acc. No. AEZ67457.1), S. scutellarioides (GenBank Acc. No. AFZ94859.1), P. vulgaris (GenBank Acc. No. AHY94892.1), O. europaea (GenBank Acc. No. AHZ31605.1), P. cablin (GenBank Acc. No. AJO53273.1), M.

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officinalis (GenBank Acc. No. CBJ23826.1), E. guttata (GenBank Acc. No. XP_012836017), S. indicum (GenBank Acc. No. XP_011077338.1). The active sites residues displayed in a

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box are the conserved amino acid residues aligned across various plant families.

Fig. 2. Phylogenetic tree of PAL protein sequences of various plants: A. rugosa (GenBank Acc. No. AF326116_1), P. vulgaris (GenBank Acc. No. AHY94892.1), S. miltiorrhiza (GenBank Acc. No. ABD73282.1), M. officinalis (GenBank Acc. No. CBJ23826.1), O. basilicum (GenBank Acc. No. KU375119), P. frutescens (GenBank Acc. No. AEZ67457.1), R. glutinosa (GenBank Acc. No. AF401636_1), O. europaea (GenBank Acc. No. AHZ31605.1), E. guttata (GenBank Acc. No. XP_012836017), S. viscidula (GenBank Acc. No. ACR56688.1), S. baicalensis (GenBank Acc. No. ADN32767.1), P. cablin (GenBank Acc. No. AJO53273.1), S. scutellarioides (GenBank Acc. No. AFZ94859.1), S. indicum (GenBank Acc. No. XP_011077338.1). Construction of the phylogenetic tree was carried out by mega 6. 0. Numbers above the branches indicate bootstrap values.

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Fig. 3. Predicted tertiary structure of ObPAL protein established by homology-based modeling (The NDN motif, Ala-Ser-Gly tripeptide and N-, C-terminal are marked red, green and yellow, respectively).

Fig. 4. SDS-PAGE analysis of ObPAL expressed in E. coli. Lane M protein molecular weight marker (MW), Lane 1 E. coli harboring empty vector, Lane 2 E. coli harboring pET2-28a(+)ObPAL construct induced by IPTG (1 mM) at 7 h.

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4000 3000 2000 1000 0 50

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Temperature (°C)

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Enzyme activity (U)

5000

7

W1= 75% FC

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W2= 50% FC

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a

W3= 25% FC

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Gene expression relative

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Fig. 5. The optima temperature analysis of recombinant ObPAL expressed in E. coli BL21. Each data represents the average of three experiments and the error bars indicate the standard deviation.

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a a

4

b

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b c

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c

c

1 0 Cul. 1

Cul. 2

Cul. 3

Fig. 6. Differential expression levels of the ObPAL gene in the three cultivar of Ocimum. basilicum under different treatments of drought stress (W1, W2, W3 are 75, 50 and 25 % FC, respectively). Different letters (a, b, c) indicated above the bar represent statistically significant difference at p≤ 0.05 (Duncan's multiple range test).

ACCEPTED MANUSCRIPT Highlights •

The sequence of PAL in Ocimum basilicum revealed a significant evolutionary affinity with that of several taxa within Lamiaceae

•

The recombinant protein obtained from cloning of PAL into pET28a (+) vector

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exhibited high PAL activity and catalyzed the conversion of L-Phe to t-cinnamic acid •

PAL manifested various transcription ratios exposed to drought stress

•

The regulation of PAL in Ocimum basilicum is possibly a cultivar- and drought stress-

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dependent mechanism

ACCEPTED MANUSCRIPT Author contributions F.K. and M.R. conceived the experiments; H.A. supervised the experiments; M.R. and F.K. analyzed the results;

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F.K. wrote the manuscript.