key: cord-0739601-nghgjtbn
authors: Srinath, Mote; Shailaja, Aayeti; Bindu, Byreddi Bhavani Venkata; Giri, Charu Chandra
title: Molecular Cloning and Differential Gene Expression Analysis of 1-Deoxy-D-xylulose 5-Phosphate Synthase (DXS) in Andrographis paniculata (Burm. f) Nees
date: 2020-11-22
journal: Mol Biotechnol
DOI: 10.1007/s12033-020-00287-3
sha: c69eb0961f867e00e34942941a9e1fae8647a53e
doc_id: 739601
cord_uid: nghgjtbn

Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1) was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 µm JA elicitation followed by 50 µm. HPLC data indicated that the transcription levels were correlated with increased andrographolide accumulation. The peak level of andrographolide accumulation was recorded at 25 μM JA (9.38-fold) followed by 50 µM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further, prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12033-020-00287-3) contains supplementary material, which is available to authorized users.

Isoprenoids are the important diverse group of bioactive compounds performing vital biological functions in plants, bacteria, and mammals. Especially in plants, they perform significant roles such as redox reactions, light harvesting, growth regulation, membrane structure, and development [1] . All isoprenoids are synthesized by two 5-carbon monomers, isopentenyl pyrophosphate (IPP), and its isomer, dimethyl allyl pyrophosphate (DMAPP). The biosynthesis of these 5-carbon monomers was achieved by the mevalonate (MVA) and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathways [2, 3] . However, a complete understanding of these highly complex biosynthetic pathways has been elucidated in only few plants. The MVA pathway is a main biosynthetic pathway in all eukaryotes, whereas the MEP pathway is the dominant source of isoprenoids in eubacteria, algae, higher plants, and protists [4] . In photosynthetic organisms, both precursor pathways are operative in different sub-cellular compartments. The MVA pathway occurs in the cytoplasm whereas the MEP pathway operates in plastids (Fig. 1) . Limited exchange of IPP and DMAPP between the plastid and cytosol was detected in several plants, but this crosstalk is not sufficient to understand the biosynthetic limitations in either pathways [5] . Isoprenoids are produced in response to external stimuli (biotic/abiotic), and/or accumulate exclusively in specialized tissues, but in small amounts due to their high degree of chemical complexities, metabolic specialization, and functional attributes [6] .

In addition, the MEP pathway is a prominent source for the effective biosynthesis of isoprenoids in plants that delivers C5 prenyl diphosphates for synthesis of monoterpenes, diterpenes, and tetraterpenes [3, [7] [8] [9] . 1-Deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) catalyzes the condensation of pyruvate and D-glyceraldehyde 3-phosphate (G3P) into DXP, a rate limiting step that controls flux in the MEP pathway [10, 11] . The role of DXS in plants and its conserved regulation among the plants signifies its importance [12] . It also suggests that the DXS expression is linked with the plant parts in a tissue-specific manner and the accumulation of the final products [13, 14] . The enhanced production of these compounds, particularly diterpene lactones can be achieved through genetic manipulation of biosynthetic pathway [15, 16] . The DXS gene has made known to be a significant target for the manipulation of terpenoids biosynthesis [17] [18] [19] [20] [21] . It is essential to detect the amino acid residues prerequisite to preserve the structure and function of DXS. Structural information is required to understand the molecular mechanisms of DXS conformational changes and their roles in catalysis until crystal structures are available [22] .

Andrographis paniculata (Family: Acanthaceae), a wellknown annual medicinal herb, is native to India [23] . This plant has been used for the treatment of common cold and flu as a traditional remedy. At one point of time, it was mentioned that the plant acted as a wonder drug during the 1919 Spanish flu pandemic akin to current COVID-19 crisis [24] . It is referred to as "Indian Echinacea" by Strong [25] . Fig. 1 Schematic representation of the MEP pathway for DL biosynthesis in Andrographis paniculata. G3P glyceraldehyde-3-phosphate, DXS 1-deoxy-D-xylulose 5-phosphate synthase, DXP 1-deoxy-Dxylulose 5-phosphate, DXR 1-deoxy-D-xylulose 5-phosphate reductoisomerase, MEP 2-C-methyl-D-erythritol 4-phosphate, DMAPP dimethylallyl diphosphate, IPP isopentenyl diphosphate, GGPPS geranylgeranyl diphosphate synthase, GGPP geranylgeranyl diphosphate, Ent-CPS Ent copalyl diphosphate synthase, GT glycosyl transferase The plant was evaluated for its various medicinal properties over the years such as anti-cancer [26] [27] [28] , anti-viral [29, 30] , cardio protecting [31] , and neuro-protectant [32] . A. paniculata is one of the main ingredients in pain killer and anti-inflammatory drugs, e.g., ParActin and ArmaForce. The medicinal attributes of this plant are due to the presence of diterpene lactones (DLs) and their derivatives [33] (Fig. 2 ). An understanding of mechanisms that regulate IPP biosynthesis in A. paniculata is of paramount importance for the production of bioactive compounds particularly DLs. Further, the regulation of DXS gene will help in the manipulation of the biosynthesis DLs as MEP pathway provides precursor IPP. However, most of the genes in the MEP pathway have not been isolated and characterized as yet in A. paniculata particularly DXS. In our previous study, we have isolated and characterized ApHMGR gene from its cytosol counterpart MVA pathway and assessed its role in the DL biosynthesis [34] .

Keeping in view significance of the MEP pathway in the biosynthesis of DLs, we report the isolation of full-length cDNA of ApDXS, a rate-limiting key gene of the MEP pathway from A. paniculata for the first time and its heterologous expression in E. coli. Prediction of conserved domains was performed to find the sequence similarities of ApDXS compared to other plant DXSs. Further, virtual docking was carried out to identify catalytic amino acids essential for Fig. 2 Important diterpene lactones present in A. paniculata. Andrographolide and its derivatives were represented in black, whereas the andrographolide analogs were represented in blue color. 3a-f 3,19-diacetyl-C-12-substituted-14-deoxy-andrographolide, 3A.1 19-tert-butyldiphenylsilyl-8,17-epoxy andrographolide, 5a-n 14α-O-(1,4-disubstituted-1,2,3-triazolyl) ester derivatives of andrographolide 17b 19-acetylated 14α-(5′,7′-dichloro-8′-quinolyloxy) derivative, 3 14β-(8′-quinolyloxy)-3,19-diol derivative, 3g 3,19-diacetyl-12-phenylthio-14-deoxy-andrographolide, 8m 3,19-(2-chlorobenzylidene) andrographolide, 3b 3,19-(3-chlorobenzylidene)andrographolide, AGS-30 3,19-(2-chloro-5-nitrobenzylidene)andrographolide interaction of the ApDXS enzyme with its substrates and inhibitors. These include N-terminal targeting sequence, a conserved thiamine diphosphate-binding site, and pyridinebinding DRAG domains. The present study also reports, comparative differential ApDXS expression pattern among different in vivo plant parts and andrographolide accumulation using in vitro adventitious root cultures in response to JA driven elicitation. One of the objectives of our comprehensive research program has been to make genomic information avalilable about andrographolide biosynthesis pathway genes in A. paniculata. The present findings will also help in understanding the role of ApDXS in DL biosynthetic pathway.

A. paniculata plants developed in vitro were used for the isolation of total RNA. A. paniculata seeds were collected from net house-established plants at Centre for Plant Molecular Biology (CPMB), Osmania University, Hyderabad, India. Seeds were surface sterilized with 0.1% (w/v) HgCl 2 for 1 min followed by washing with sterile distilled water and inoculated onto the Murashige and Skoog's (MS) medium [35] . The MS medium was fortified with 3% (w/v) sucrose and 0.8% (w/v) agar (Fisher Scientifics, Mumbai, India). The seeds were then incubated under dark for germination.

Approximately, 100 mg leaves were collected from 2-monthold in vitro grown plants using a sterile scalpel and placed in −20 °C for 5-10 min. The leaves were then pulverized to a fine powder in liquid nitrogen using pre-cooled sterile mortar and pestle. 1.0 ml of RNAiso Plus (DSS Takara Bio India Pvt. Ltd. New Delhi, India) was added immediately. Total RNA was isolated following Trizol method and converted to cDNA using DSS Takara Prime Script First-Strand cDNA Synthesis kit (DSS Takara Bio India Pvt. Ltd. New Delhi, India) as per manufacturer's instructions. cDNA was used as a template for further polymerase chain reactions (PCR).

DXS protein sequences from other closely related plants were obtained from NCBI. Conserved regions among the sequences were identified by multiple sequence alignment using the Clustal Omega tool. The primers based on highly conserved regions were designed using the primer3 tool ( Table 1 ). The primers were designed with the help of an Oligo Analyzer web tool by avoiding primer dimer and hair pin formation. The selected primers were procured from ReGene Biologics Pvt. Ltd., Hyderabad, India. PCR reactions were carried out with gradient PCR machine (Eppendorf AG, Hamburg, Germany) using cDNA as a template. The DreamTaq DNA polymerase (Thermo Scientific, USA) was used for the amplification of a full-length gene. The PCR reaction was as follows: initial denaturation -94 °C for 2 min; 35 cycles of denaturation -94 °C for 1 min; annealing -62.3 °C for 1 min; extension -72 °C for 2.3 min; final extension -72 °C for 5 min; and rapid cooling -4 °C.

The PCR amplicon was gel eluted and purified using a FavorGen PCR cleanup kit (FavorGen, Taiwan) as per manufacturer's instructions and given for sequencing at ReGene Biologics Pvt. Ltd., Hyderabad, India. The sequence obtained was submitted to NCBI and subjected to bioinformatics analysis using the Clustal Omega and ESpript tools to find the sequence comparison with other plant DXS genes.

The full-length ApDXS gene was cloned into the pET 32 ( +) expression vector. The primers were designed with two separate restriction sites ( Table 2 ). The PCR products cut with restriction enzymes were ligated with linearized pET 32 ( +) vector. The cloned ApDXS gene was transformed into E. coli Rosetta competent cells. Recombinant colonies were identified by screening for ampicillin resistance. The transformed recombinant colonies were 

The different tissues such as leaf, stem, and root were collected from 2-month-old in vitro grown plants. The tissue was divided into two parts. One part was used for tissue-specific expression profiling of ApDXS gene. Another part was used for estimation of andrographolide content by the High-Performance Liquid Chromatography (HPLC). Approximately, 100 mg of fresh tissue was used for total RNA isolation by Trizol method and converted to cDNA as mentioned earlier. The primers were designed specifically for qRT-PCR using full-length ApDXS gene sequence with product size 250 bp ( Table 3 ). The qRT-PCR was performed in Applied Biosystems 7500 Fast PCR (Applied Biosystems, Foster City, USA) machine using DyNAmoColorFlash SYBR Green qPCR master mix (Thermo Fisher Scientific, USA). The gene expression was calculated by 2 − ΔΔCt (cycle threshold) method. The normalization was carried out by Actin gene [36] .

About 50 mg of dried plant tissue powder from different plant parts was subjected to methanol extraction as per Zaheer and Giri [37] with minor modifications. Dried powder was added to 5 m of HPLC grade methanol (Fisher Scientifics, India) and incubated at room temperature for 24 h followed by 1 h sonication in Ultrasonic Cleaning Bath (Spectralabs Instruments Pvt. Ltd, Mumbai, India). The methanol extract was filtered through Whatman No.41 filter paper first and then through 0.2 µm Millex GV Durapore PVDF filter (Merck, Germany). The filtered sample (500 µl) was used for analysis in the Waters HPLC system (Waters, USA) using a C18 column. The methanol (100%) was used as an isocratic solvent system. The samples were run for 15 min with 20 µl injection volume and 1 mL/min flow rate at 25 °C. The pressure was maintained in the range of 2000-6000 psi. The andrographolide content was detected with the PDA detector. The authentic andrographolide (98%) procured from Sigma-Aldrich (USA) was used as a standard. Andrographolide content was estimated by calculating peak areas and comparing with the authentic standard.

The seeds collected from net house-grown A. paniculata plants were germinated in vitro as mentioned earlier. The cotyledon explants from these in vitro germinated plantlets were inoculated onto MS semisolid medium containing 1.0 mg/l naphthalene acetic acid (NAA) for the induction of adventitious roots. The three-week-old actively growing adventitious roots were subsequently transferred to the same MS + NAA medium supplemented with different concentrations of JA viz.1.0, 10, 25, 50, and 100 µM as per Zaheer and Giri [37] . The growth indices (GI) of the elicited adventitious roots were conducted by harvesting the adventitious roots and measuring fresh and dry weight for a duration of TCT CCT TGC TCA TCC TGT CC  200 bp  6 Actin-qRT-PCR reverse AAG AAC TAT GAG CTG CCC GA 4 weeks. The adventitious roots harvested were weighed, dried, and powdered using mortar and pestle. The dried powder (50 mg) was subjected to methanol extraction as mentioned earlier. The standard andrographolide procured from Sigma-Aldrich, USA was prepared in 1.0 mg/mL concentration with HPLC grade methanol and estimated in samples by comparing its UV absorbance and retention time.

The induction of adventitious roots and elicitation experiments were conducted thrice with minimum of 6 replicates each, and the data were expressed as mean ± SE.

The differential expression analysis of ApDXS along with ApHMGR was investigated by qRT-PCR in A. paniculata JA-elicited in vitro adventitious root cultures. The RNA was isolated by Trizol method from the adventitious root culture samples elicited with different concentrations of JA. The RNA was quantified by NanoDrop (Eppendorf, Humbarg, Germany) and converted to cDNA. Consequently, the cDNA of all the samples was used for qRT-PCR reactions in 7500 Fast Real-Time PCR (Applied Biosystems, USA) using the DyNAmoColorFlash SYBR Green qRT-PCR kit (Thermo Scientifics, USA). The actin gene was used as an endogenous control for normalization of gene expression. The relative gene expression was calculated following the 2 − ΔΔCt (cycle threshold) method [36] . The qRT-PCR analysis was performed with 3 experimental replicates.

The 3D model of ApDXS was generated using the Swiss model web server (https ://swiss model .expas y.org/). The E. coli DXS (SMTL ID: 2o1s.1) was used as a template which has 47% sequence identity with ApDXS. The 3D model was further validated using the Structure Analysis and Verification Server (Saves) v5. 0 (https ://servi cesn.mbi.ucla.edu/ SAVES /), a metaserver (consisting Procheck, Verify 3D, Errat, etc.) for protein structures during and after model refinement. The evaluation of amino acid residues present in the most favored regions of the designed 3D model of ApDXS was checked using RAMPAGE Ramachandran plot assessment web tool (http://mordr ed.bioc.cam.ac.uk/~rappe r/rampa ge.php).

The DXS catalyzes the condensation of pyruvate and G3P to produce DXP using thiamine pyrophosphate (TPP) and Mn2 + . Hence, the structures of pyruvate 

The gene-specific primers designed from the conserved regions of other plant DXSs, amplified a partial ApDXS sequence of 1014-bp initially (GenBank: MG271750.1).

Later the full-length cDNA of ApDXS (NCBI Accession Number-MG271749.1) of 2,453-bp containing 322-bp 5′ untranslated region (UTR), 2076-bp open reading frame (ORF) and 55-bp 3′ UTR was amplified (Fig. 3) . It encoded a protein of 691 amino acids with a deduced molecular weight of 74.4 K Da and a theoretical pI of 6.01.

According to restriction site analysis, the ORF of the ApDXS gene of A. paniculata was inserted into expression vector pET-32a ( +) between Not I and Kpn I restriction sites. The verification of recombinant prokaryotic expression vector in IPTG-induced bacteria through SDS-PAGE showed a specific band of 74.4 KDa-expressed protein (Supplementary File-1. S1).

The qRT-PCR results revealed that ApDXS gene was expressed in all examined tissues, namely leaves, stems, and roots. ApDXS gene expression levels were significantly higher (16-fold) in leaves followed by stem and roots. The expression level of ApDXS gene in stem was observed to be fourfold higher than that in roots (Fig. 4) .

The highest level of ApDXS gene expression was observed in 25 µM JA treatment followed by 50 µM JA in all week (Fig. 5a) . The highest HMGR expression was observed in (Fig. 5b) . Adventitious root biomass was enhanced only at 3 rd week in 50 μM JA (Fig. 6a) . The highest level of andrographolide accumulation was recorded at 25 μM JA (9.38-fold) followed by 50 µM JA (7.58-fold) treatment. The accumulation was more profound in the case of 25 μM JA treatment (Fig. 6b) . All the experiments were resulted with ≤ 0.05 standard error. Raw data of differential expression analysis of ApHMGR and ApDXS in JAelicited in vitro adventitious root cultures of A. paniculata 

The nucleotide sequence of ApDXS submitted to the NCBI GenBank database and was assigned with the Accession No. MG271749. 1 (AAS89341.1, 82.99% ). The comparison of the deduced amino acid sequence of ApDXS and the above organisms revealed highly conserved regions (Fig. 7) . A highly consensus fingerprint structural transketolase TPP-binding motif GDG-X26-NDN was found in residues 188-217. A cluster of amino acids known to be (glyceraldehyde-3-phosphate) substrate-binding motifs (GGHLGS, VSIHVVTEKG, KPFCAIYSSFLQ, AND AMDRAGLV-GADGP) were identified in the ApDXS proteins, corresponding to the residues 84-90, 327-336, 437-448, and 468-480, respectively. Towards the N-terminal, another highly conserved target sequence VAIHYVFNA was found at residues 98-106. The N-terminal ends of all plant DXSs were not identical, but share a common feature of the chloroplast transit peptide (cTP) sequence. The predicted cTP cleavage site (EYFSEKPPTP) and was positioned between amino acids 37 and 46. TheApDXS was cleaved between 'S' and 'E' at residues 40 and 41 in the processing sites, and the putative mature ApDXS was deduced. In addition, a conserved landmark pyrimidine-binding motif DRAG was found at residues 470-473, transit peptide RRF at 409-411 along with essential catalytic regions GIAEQHAVTF (418-427), SSFLQRAYD (444-452), and PSCFRYPRGNG (522-532) (Fig. 7) .

The 3D structure of ApDXS generated by the Swiss model server was represented in ribbons (Fig. 8) . Structural assessment through Ramachandran plot revealed 98% expected amino acid residues in the favored and 2% in the allowed regions with 92% structural reliability. The Ramachandran plot can be found in the Supplementary material-1 (S.2). The in silico molecular docking studies revealed that pyruvate and G3P, substrates of DXS, were binding at Region-I (VIGDGAMIAGQAYEAMNNAGYLDSDMIVILND) and Region-II (AMDRAGLVGADGP), respectively. The co enzyme TPP was observed to bind at Region-II more efficiently than at Region-I. Clomazone, ketoclomazone inhibitors, and intermediate compound IPP were effective to bind Region-II. The other inhibitor fluoropyruvate was observed to bind more efficiently at Region-I than to a Region-II. Among all the molecules, methyl acetyl phosphonate was observed as a week binder at both the regions. Binding of molecules to their respective binding sites and the hydrogen bonds between molecules and DXS active sites can be established in the Supplementary material-file1 S.3. Binding energies and H-bond score and interacting amino acid residues are given in Table 4 .

The pyruvate was bound to its substrate-binding pocket by forming hydrogen bonds with Ser (247 position), Asn (307), and Asp (304) whereas G3P was interlinked with Arg (252) by one H-bond, Asp (304, 310) via two H-bonds, Asn (248, 307) with two hydrogen bonds, and Ser (247) and Leu (251) with one H-bond. The TPP was bound to its pocket via 7 H-bonds with Leu (254), Arg (255), Lys (66), His (306), Arg (337 and 73), and Glu (95). The clomazone has formed only single H-bond with each of Arg (249) and Asn (307), whereas the ketoclomazone was found to have two H-bonds with each of Arg (249) and Ser (247), respectively. The IPP formed 2 hydrogen bonds with each of Arg (255) and Asp (304), and one H-bond with Asn (307). The fluoropyruvate was also observed to form single hydrogen bonds with each of Leu (228), Ala (226), and Thr (225). Methyl acetyl phosphonate was interlinked with DXS protein at Asn (307) by forming a single hydrogen bond.

Despite the intensive study of genes and proteins contributing for diterpene biosynthesis in A. paniculata plant, a meager information is available for the isolation and characterization of the MEP pathway genes. However, in our laboratory, we have ongoing comprehensive research programs to decipher both MVA and MEP pathway genes using genomic and proteomic approaches [34, 38] . In the present study, we have isolated, cloned, and carried out in silico functional characterization of DXS gene from A. paniculata for the first time. Further, we have done its tissue-specific expression in different plant parts. The effect of JA elicitation on the expression levels of rate-limiting enzymes HMGR and DXS from MVA and MEP pathways, respectively, was carried out in JA-elicited in vitro adventitious root cultures correlating with andrographolide production to understand the synergy within elicitation vis-à-vis the cross talk between metabolic networks.

Although the DXS gene from other plants has been isolated and analyzed, isolation of DXS gene from A. paniculata was not reported earlier. Here, the successful isolation of full-length cDNA of DXS gene from A. paniculata and its heterologous expression in E. coli provides a basis for a genetic resource for its exploitation. In earlier studies, the cloning, characterization, and overexpression of DXS gene showed increased abietane diterpenes and steviol glycosides synthesis in Salvia sclarea and Stevia plants, respectively Fig. 7 Identification of conserved domains and motifs in ApDXS using Multiple Sequence Alignment with other known plant DXS proteins. Red star-highly conserved catalytic residues; green triangles-presumed transit peptide cleavage site; hollow circles-Glyceraldehyde-3-phosphate binding sites; pink star-Transketolase TPP-binding domain; blue star-N-terminal highly conserved target sequence; green star-essential catalytic regions. The figure is prepared using the ESpript web server (Color figure online) ◂ [18, 21] . ApDXS encoded a protein of 691aa long which is in the range of plant DXSs. In general, the plant DXS protein ranged from 691 to 738aa long [19, 20, [39] [40] [41] . The deduced amino acid sequence upon BLAST showed high similarities with plant DXS super family genes, indicating its close proximity to other functional DXS proteins. Further, the main objective of our study was to generate and provide the genomic information of DXS as a genetic resource from A. paniculata for the first time, and it fulfills our goal giving an indication that ApDXS gene is active in transcription stage. Here, in our study, qRT-PCR was employed to investigate the expression profiles of ApDXS gene in different tissues, including leaves, stems, and roots. Gene expression profiling revealed differences in expression pattern as observed in Populus trichocarpa and Artemisia annua plants earlier.

The tissue-specific expression of DXS suggested its elevated expression levels in leaves compared to the stem and roots [19, 42] . Our initial studies have shown that the andrographolide content was highest in the leaves, followed by stem and roots. High-expression levels of ApDXS in leaves were observed similar to findings in other plants such as Arabidopsis thaliana [43] , Amomum villosum [44] , Aquilaria sinensis [45] , and Tripterygium wilfordii [46] . In our previous report, we found ApHMGR of the MVA pathway was highly expressed in roots than the stem and leaves. It was identified that HMGR expression was higher in roots (2.5 fold) followed by stem (twofold) and leaf tissues (1.5 fold) [34] . Hence, we could draw a conclusion that DXS from MEP and HMGR from MVA pathway have prominent roles for the production of andrographolide and other DLs. Therefore, the tissue-specific expression could be used for the manipulation of biosynthetic pathways localized in these plant parts for the biosynthesis of specific secondary metabolites.

The findings on tissue-specific expression imply that, the ApDXS gene expression was relatively low in the roots in comparison to that in the leaves in vivo. Although its expression is low in roots, the potential of in vitro adventitious root cultures cannot be apparently overruled for andrographolide biosynthesis. In order to deduce the underlying molecular mechanism and regulation of the rate limiting genes of MEP and MVA pathways for the biosynthesis of andrographolide, we categorically carried out in depth study involving JA mediated elicitation using in vitro adventitious root cultures. Differential expression of ApDXS from MEP pathway and ApHMGR from MVA pathway was studied. The mechanism of action of JA elicitation has already been discussed earlier [47] . The influence of both in vitro culture conditions and JA elicitation was considered. The relative expression levels of the ApDXS and ApHMGR genes in elicited in vitro adventitious root cultures showed different degrees of gene expression patterns over the course of JA treatments for 4 weeks. Further, a significant increase in andrographolide accumulation was recorded after JA elicitation treatment. The expression of ApDXS increased gradually from the second week, whereas ApHMGR was enhanced from first week to fourth week in all the concentrations. The elicitation with JA in all concentrations except 100 µM (at 3rd week) did not promote biomass enhancement of adventitious roots. Although biomass was not enhanced, biosynthesis of andrographolide was observed to be elevated from HPLC and qRT-PCR experiments. The results of these experiments indicated that ApDXS and ApHMGR played significant roles in the biosynthesis of DLs (andrographolide) in JAelicited adventitious roots. The transcription levels of both the genes in JA-treated adventitious roots were enhanced gradually with increased JA concentrations and maintained levels well above compared to untreated control. Maximum expression level of ApDXS and ApHMGR was observed at 25 µM followed by 50 µM of JA. Compared to ApDXS expression, ApHMGR gene expression was low but significantly enhanced than untreated controls. Upregulation of ApHMGR was started after the first week and gradually increased till the fourth week in all the concentrations. Evidently, the higher expressions of ApDXS and ApHMGR genes at 25 µM and 50 µM with higher andrographolide synthesis indicated the crucial role of these two genes and the synergy between MVA/MEP pathways. Singh et al. [48] reported that untreated control, in vitro adventitious root cultures of A. paniculata have shown high-expression levels of MVA pathway genes (HMGR1/2) and low expression of MEP pathway genes (DXS1/2 and HDR1/2). However, in our study, an external stimulus JA not only enhanced the MEP pathway-related gene expression, but also significantly influenced MVA pathway genetic. Several studies from plants, e.g., maize, rice, barley, and Medicago roots [49, 50] , Ginkgo biloba [51] , Picea abies [52] , and Pinus densiflora [53] , have shown that, DXS gene displayed a consistent expression pattern when subjected to external stimuli (electors: MeJA, MeSA, Chitosan, Arbuscular mycorrhizal fungi, etc.) for isoprenoid-derived secondary metabolites.

A search with the BLASTp program indicated that the ApDXS was highly homologous to DXS sequences from other plants signifying its lineage to the DXS gene and protein family. 3D model of ApDXS was generated, validated, and reported earlier by Srinath et al. [54] and was used for in silico docking studies. Our study finds significance, since plant DXS has no inhibitor-bound co-crystal structure known which makes a structure-based ligand optimization difficult [55, 56] . The ever-demanding need for the regulation of biosynthetic pathway for the enhancement of DLs requires more efforts to identify new molecular scaffolds in ApDXS. Studies of these enzyme-substrate/ligand interactions are relatively more important in molecular interventions to find the regulatory role of DXS and subsequent MEP pathway engineering for the enhanced biosynthesis of DLs [57, 58] . In silico molecular docking analysis brought in focus some important interactions of ApDXS enzyme to its ligands. In turn, these evidences could guide a rational design of new potential inhibitors for DXS. The docking studies from the present work in A. paniculata that revealed Arg (249, 252, 255), Asn (307), and Ser (247) amino acid residues are important and play a vital role in forming interactions. These amino acid residues and their interactions are expected to provide useful insights for the in vitro enzyme inhibition studies.

Our present finding may be attributed to the JA mechanism of action in inducing genes and transcription factors in adventitious root cultures of A. paniculata [38, 47] . Such observation further highlighted the combo action of in vitro adventitious root cultures, and JA elicitation can contribute towards understanding the gene expression pattern and biosynthesis of andrographolide and other DLs. Previous studies on elicitation and identification of gene regulation pattern for secondary metabolite production give credence to the present findings [59] [60] [61] [62] .

Although the MEP pathway was discovered two decades ago, the key genes are not fully characterized yet. Isolation of the genes responsible for the rate-limiting enzymes is a positive step towards pathway manipulation. Due to the central role of this MEP pathway, identification of the major regulatory steps is of paramount importance for the growth and development of plants and their exploitation in human health care. In conclusion, we isolated full-length gene-encoding ApDXS which is involved in the biosynthesis of DLs in A. paniculata. Heterologous expression of the ApDXS was carried out in E. coli. The present study with the isolation and characterization of ApDXS contributed towards generating genetic resources in A. paniculata for its further exploitation. We analyzed the differential expression patterns of ApDXS in various tissues and identified the differential expression levels of ApDXS along with ApH-MGR under JA elicitation using in vitro adventitious root cultures. The correlation between differential gene expression and andrographolide biosynthesis further confirmed that ApDXS and ApHMGR, the key regulatory enzymes of MEP and MVA pathways, were responsible for the biosynthesis of DLs in A. paniculata. These observations are useful in studying how the plastidial MEP pathway complements the cytosolic MVA pathway in different plant parts such as leaves, stem, and roots for producing the C5 units of andrographolide and other DLs. The given information on gene isolation, heterologous expression, and differential gene expression from our study will contribute to the understanding of andrographolide biosynthesis using elicitation of in vitro cultures as an experimental model. This study provides objective to explore possibilities for manipulating DXS and HMGR enzymes in onjunction with other downstream enzymes specific for the diterpene lactone biosynthesis. The present study indicated that the JA has substantially influenced the expression of DXS and HMGR from both MVA and MEP pathways and ultimately controlled the regulation of andrographolide biosynthesis in A. paniculata. This finding also suggests that the over expression of isolated gene and application of external stimuli such as JA can modulate the biosynthetic pathways to enhance the production of andrographolide and will be advantageous in pathway engineering using in vitro culture systems. The state-of-theart bioinformatics and in silico docking findings from the present study have added valuable information about the essential structural attributes of the ApDXS protein needed for its function until experimental models become available in A. paniculata. Further, the present study with the isolation and characterization of ApDXS contributed towards generating genetic resources in A. paniculata for its manipulation.

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Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

Medically useful plant terpenoids: Biosynthesis, occurrence, and mechanism of action

Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life

Network analysis of the MVA and MEP pathways for isoprenoid synthesis

Biosynthesis of plant isoprenoids: perspectives for microbial engineering

Plant isoprenoid biosynthesis via the MEP pathway: in vivo IPP/ DMAPP ratio produced by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase in tobacco BY-2 cell cultures

The potential of the mevalonate pathway for enhanced isoprenoid production

Volatile terpenoids: multiple functions, biosynthesis, modulation and manipulation by genetic engineering

The methyl erythritol phosphate pathway to isoprenoids

Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound

Breaking new ground in the regulation of the early steps of plant isoprenoid biosynthesis

Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize

Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants

Engineering the methylerythritol phosphate pathway in cyanobacteria for photosynthetic isoprene production from CO 2

Creating plant molecular factories for industrial and nutritional isoprenoid production

Bioinformatics study of 1-deoxy-d-xylulose-5-phosphate synthase (DXS) genes in Solanaceae

High yield of bioactive abietane diterpenes in Salvia sclarea hairy roots by overexpressing cyanobacterial DXS or DXR genes

Evaluation, characterization, expression profiling, and functional analysis of DXS and DXR genes of Populus trichocarpa

The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide

Overexpression of SrDXS1 and SrKAH enhances steviol glycosides content in transgenic Stevia plants

X-ray crystallography-based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate

Distribution of andrographis species in different districts of Andhra Pradesh

A double-blind study with a new monodrug Kan Jang: Decrease of symptoms and improvement in the recovery from common colds

Andrographolide (AP1) inhibits cholangiocarcinoma cell invasion in vitro model. Hepatobiliary Surgery and Nutrition

Andrographolide inhibits cholangiocarcinoma cell migration by down-regulation of claudin-1 via the p-38 signaling pathway

Advances and challenges in developing andrographolide and its analogues as cancer therapeutic agents

A proteomic analysis of the anti-dengue virus activity of andrographolide

Design, synthesis and discovery of andrographolide derivatives against Zika virus infection

Andrographolide Protects against adverse cardiac remodeling after myocardial infarction through enhancing Nrf2 signaling pathway

Neuroprotection of andrographolide against microglia-mediated inflammatory injury and oxidative damage in PC12 neurons

Identification of bioactive diterpenoid lactones using nuclear magnetic resonance from Andrographis paniculata

Isolation, characterization and in silico analysis of 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) gene from Andrographis paniculata (Burm. f) Nees

A revised medium for rapid growth and bio assays with tobacco tissue cultures

Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells

Enhanced diterpene lactone (andrographolide) production from elicited adventitious root cultures of Andrographis paniculata

Proteome analysis and differential expression by JA driven elicitation in Andrographis paniculata (Burm. f.) Wall. ex Nees using Q-TOF-LC-MS/MS

Molecular cloning and expression of 1-deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoisomerase in Dendrobium officinale

Cloning and prokaryotic expression of 1-deoxy-Dxylulose-5-phosphate synthase (DXS) from Lepidium apetalum

Partial sequence isolation of DXS and AOS genes and gene expression analysis of terpenoids and pyrethrin biosynthetic pathway of Chrysanthemum cinerariaefolium under abiotic elicitation

Molecular characterization of the 1-deoxy-D-xylulose 5-phosphate synthase gene family in Artemisia annua

1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants

Characterization and functional analysis of the genes encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase and 1-deoxy-D-xylulose-5-phosphate synthase, the two enzymes in the MEP pathway, from Amomum villosum Lour

Molecular cloning and characterization of three cDNAs encoding 1-deoxy-D-xylulose-5-phosphate synthase in Aquilaria sinensis

Molecular cloning and characterization of DXS and DXR genes in the terpenoid biosynthetic pathway of Tripterygium wilfordii

Chemical elicitors versus secondary metabolite production in vitro using plant cell, tissue and organ cultures: recent trends and a sky eye view appraisal

Anti-cancer labdane diterpenoids from adventitious roots of Andrographis paniculata: augmentation of production prospect endowed with pathway gene expression

Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with accumulation of the 'yellow pigment'and other apocarotenoids

Two distantly related genes encoding 1-deoxy-d-xylulose 5-phosphate synthases: Differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots

Identification of class 2 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis

Functional identification and differential expression of 1-deoxy-D-xylulose 5-phosphate synthase in induced terpenoid resin formation of Norway spruce (Picea abies)

Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-D-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes

Characterization of 1-deoxy-D-xylulose 5-phosphate synthase (DXS)

Nees: A in silico appraisal

A high-throughput screening assay for simultaneous selection of inhibitors of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate synthase (DXS) or 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR)

Inhibitors of the herbicidal target IspD: Allosteric site binding

Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway

Antimicrobial N-(2-chlorobenzyl)-substituted hydroxamate is an inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase

Jasmonic acid and methyl dihydroJasmonate enhance saponin biosynthesis as well as expression of functional genes in adventitious roots of Panax notoginseng FH Chen

Expression analysis of ginsenoside biosynthesisrelated genes in methyl Jasmonate-treated adventitious roots of Panax ginseng via DNA microarray analysis. Horticulture, Environment, and Biotechnology

Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng

Response of bioactive metabolite and biosynthesis related genes to methyl JAsmonate elicitation in Codonopsis pilosula

The authors acknowledge the financial support