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. 2018 Oct 6;25(1):47–57. doi: 10.1007/s12298-018-0612-6

Terpene profiling, transcriptome analysis and characterization of cis-β-terpineol synthase from Ocimum

Atul Anand 1,2,#, Ramesha H Jayaramaiah 1,#, Supriya D Beedkar 2,3, Bhushan B Dholakia 1, Santosh G Lavhale 1, Sachin A Punekar 4, Wasudeo N Gade 3, Hirekodathakallu V Thulasiram 2,5, Ashok P Giri 1,✉
PMCID: PMC6352525  PMID: 30804629

Abstract

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, β-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-β-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches.

Electronic supplementary material

The online version of this article (10.1007/s12298-018-0612-6) contains supplementary material, which is available to authorized users.

Keywords: Agro-infiltration assay, Metabolite profiling, Ocimum, Terpenes, Terpene synthase, Transcriptome

Introduction

Plants have evolved specialized secondary metabolite biosynthetic pathways for the synthesis of structurally and functionally complex small molecules, which are essential for their survival. In plants, accumulation pattern of the secondary metabolites is complex as it changes in the tissue- and organ-specific manners. Secondary metabolites are crucial in the protection of plants against biotic and abiotic stresses. These molecules also play crucial role in attraction of pollinators and frugivores; adaptation under given environmental conditions and communications (Dixon 1999). Terpenes are major naturally occurring organic compounds present in all forms of living systems. Two simple five-carbon building blocks [isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP)] are utilized in formation of isoprenoids. This isoprenoids synthesis is divided into four steps, which takes place either in cytosol (Mevalonic acid pathway) (McGarvey and Croteau 1995; Bach 1995) and/or plastids (Methyl erythritol pathway) (Rohmer et al. 1993; Sprenger et al. 1997; Eisenreich et al. 1998): (a) production of the five carbon (C5) monomers IPP and DMAPP; (b) condensation of C5-units (IPP and DMAPP) into geranyl diphosphate (GDP), farnesyl diphosphate (FDP) and geranylgeranyl diphosphate (GGDP); (c) cyclization and rearrangement of carbon backbone to form different forms of terpenes and (d) tailoring and downstream functional group modifications of the terpene skeleton. The compartmentalization of two biosynthetic pathways in plants that leads to the formation of DMAPP/IPP is not absolute, because in several cases one metabolite can exchange between these two pathways (Burke and Croteau 2002; Thiel and Adam 2002). Terpene synthases (TPS) catalyze biosynthesis with high regio- and stereo-chemical precision in a cascade of complex reactions involving highly reactive carbocationic intermediates that undergo a sequence of reactions such as cyclizations, alkylations, rearrangements, deprotonations and hydride shifts (Cane 1985; Croteau 1987; Cane 1990; Lesburg et al. 1998; Davis and Croteau 2000; Poulter 2006; Thulasiram and Poulter 2006; Tantillo 2011). Terpenes are classified on the basis of number of five carbon units present in the core structure viz. single isoprene unit (C5) are called hemiterpenes, two isoprene units (C10) are called monoterpenes and three isoprene unit (C15) called sesquiterpenes. Terpene derivatives are also used for the production of numerous pharmaceuticals including taxol (anti-cancer drug) (Jennewein and Croteau 2001) and artemisinin (anti-malarial drug) (Rodriguez-Concepion 2004).

Monoterpenes consist of two isoprene units with the molecular formula C10H16 and are present in secretory tissues such as oil glands of higher plants, insects, fungi and marine organisms. These compounds are widely used in flavor and fragrance industry, due to their characteristic odor. Monoterpenes include three subtypes: acyclic (myrcene, geraniol, linalool), monocyclic (limonene, α-terpineol and terpinolene) and bicyclic (α-pinene, sabinene and camphor). Monoterpenes are biosynthesized from geranyl pyrophosphate (GPP) catalyzed by monoterpene synthase. Several monoterpenes have different medicinal properties including antibacterial, antifungal, antioxidant and anti-cancerous (Garcia et al. 2008). Generally, monoterpenes are synthesized in plastids through the MEP pathway in plants whereas in other higher organisms and in yeast, MVA pathway is used (Hampel et al. 2005). Ocimum is a genus of aromatic plants that belongs to the Lamiaceae family. Members belonging to Ocimum genus are known to synthesize and accumulate various secondary metabolites including terpenes in the secretory and capitate peltate trichomes. Trichomes are located on the aerial parts of these plants and are important for interaction with surrounding by releasing volatiles. The metabolite fraction of Ocimum is mainly composed of mono- and sesquiterpenes, along with phenylpropanoids, which impart them medicinal properties, as well as flavor and taste. Here, we describe terpene profiling in six different tissues from five Ocimum species, which revealed species-specific distribution of mono- and sesqui- terpenes in different Ocimum species. These five species were selected on the basis of their distribution in phylogenetic analysis. Transcriptome profiling of leaf, inflorescence and stem tissue from O. gratissimum, O. tenuiflorum and O. kilimandscharicum revealed 38 putative TPS transcripts, which might be involved in biosynthesis of mono-, sesqui- and di-terpenes. Expression analysis of three putative TPSs (TPS1, TPS2 and TPS3) in different tissues of Ocimum species revealed significant expression variation in species-specific manner. Further, functional characterization of TPS1 along with Agrobacterium-mediated transient over-expression established its role as a cis-β-terpineol synthase.

Materials and methods

Chemicals and plant materials

All chemicals were purchased from Sigma-Aldrich (Sigma Chemical Co., USA), unless stated. Ocimum plants belonging to five species viz. O. gratissimum L. I and II (Og I and Og II), O. tenuiflorum I and II (Ot I and Ot II), O. kilimandscharicum Gürke (Ok), O. americanum (Oa) and O. basilicum I, II, III and IV (Ob I, Ob II, Ob III and Ob IV) were grown in a greenhouse at 25–28 °C with ~ 35–40% humidity and 16/8 h light and dark periods. All Ocimum plants species and types within species were phenotypically confirmed by taxonomist and voucher specimens of these the plants were submitted in the herbarium of Botanical Survey of India, Pune. All the samples were collected from 3 to 4 months old plants, immediately frozen in liquid nitrogen and stored at − 80 °C until further use. Fresh tissues were used for metabolite extraction and analysis.

Metabolite analysis from different Ocimum species

Metabolite analysis was carried out in six different tissues including young leaves (YL; top whorl), mature leaves (ML; third whorl), inflorescence (I), flower (F), stem (S) and root (R) from five Ocimum species. Extractions were performed by dichloromethane (DCM) extraction method, as reported previously (Ramesha et al. 2016; Anand et al. 2016). Briefly, fresh tissues (5 g each) from different plant parts were harvested separately and immediately soaked in 50 mL DCM for 20 h at 28 °C. The combined organic phase was kept at − 20 °C for 2 h (for lipid precipitation) and filtered. The contents were dried, weighed, re-dissolved in 2 mL DCM and subjected to GC–FID and GC–MS analyses. Each sample consisted of 3 biological replicates with triplicates of each of them. GC–FID analyses were carried out on an Agilent 7890A instrument equipped with a hydrogen flame ionization detector and HP-5 capillary column (30 m × 0.32 mm × 0.25 µm, J and W Scientific, USA). Nitrogen was used as the carrier gas at a flow rate of 1 mL/min. The column temperature was raised from 70 °C to 110 °C at 2 °C min−1, then raised to 180 °C at 3 °C min−1 and finally at 10 °C min−1 raised to 220 °C and held for 2 min. Injector and detector temperatures were 230 °C and 235 °C, respectively. GC–MS was carried out with an Agilent 5975C mass selective detector interfaced with an Agilent 7890A gas chromatograph using an HP-5 MS capillary column with helium as the carrier gas and using above-mentioned conditions. Compounds were identified by co-injection studies, comparing the retention time and mass fragmentation pattern with those of reference compounds and also compared acquired mass spectra and retention indices with those of NIST/NBS and the Wiley mass spectral library (software version 2.0, Dec. 2005).

RNA isolation and transcriptome sequencing

Total RNA was extracted from the different tissues using Spectrum™ Plant Total RNA Isolation Kit (Sigma Chemical Co., USA). For transcriptome analysis, total RNA of leaf, inflorescence and stem tissues from Og, Ot and Ok were mixed and preceded with library preparation. Transcriptome library was constructed according to the Illumina TruSeq RNA library protocol outlined in “TruSeq RNA Sample Preparation Guide” (Part # 15008136; Rev. A; Nov 2010) (Genotypic Pvt. Ltd., India) and as per the methodology of Krithika et al. (2015).

Sequence and phylogenetic analysis

The sequence analyses were carried out with Bioedit software (Ibis Biosciences, USA) and CLUSTALW2 program (www.ebi.ac.uk/Tools/msa/clustalw2/). ExPASy translate tool was used for Nucleotide sequences translation (http://web.expasy.org/translate/) and identical and similar amino acid residues were marked using BOXSHADE 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Phylogenetic analysis was performed using TPS1 (MH925775), TPS2 (MH925776) and TPS3 (MH925777), with other plant based TPS. Multiple sequence alignment was carried out with ClustalX 2.1 (http://www.clustal.org/clustal2/) and the phylogenetic tree was reconstructed with MEGA6 (Tamura et al. 2013) using the neighbour-joining method with 1000 bootstrap interactions.

Semi-quantitative and quantitative RT–PCR

The DNase-treated RNA (4 µg) was used for cDNA preparation in a 20 µL reaction using SuperScript™ III reverse-transcriptase system (Thermo Scientific). For semi-quantitative PCR, 2 µg DNase-treated RNA, each of young and mature leaves were mixed together and cDNA was synthesized as described. Semi-quantitative RT–PCR (sqRT–PCR) was performed for all three TPSs in 10 µL reaction consisting of 5 µL of 2X Jumpstart readymix, 1 µL each of 10 µM gene specific forward and reverse primers, 1 µL of cDNA optimized with endogenous control (18S rRNA) and nuclease free water was added to make up a volume of 10 µL. PCR was carried out for 30 cycles and the amplified products were run on 1% agarose gel. Quantitative RT–PCR (qRT–PCR) was performed by using SYBR Green chemistry. A typical reaction consisted of 5 µL of SYBR Green master-mix, 0.5 µL each of forward and reverse primer (10 µM) and 1 µL of diluted cDNA (1:2) with nuclease-free water added to make up a volume of 10 µL. For qRT–PCR reactions, elongation factor-1 (EF1) was used as an endogenous control and the reactions were carried out in triplicates for 3 biological replicates in the 7500 Fast Real Time PCR System (Thermo Scientific). Annealing temperature was kept at 58 °C and cycling conditions were kept as per the manufacture’s instruction. Primer sequences are as given in (Supplementary Table S1).

Cloning and in vivo expression analysis of TPS1

By comparing known TPS with de novo assembled RNA-Seq Ocimum transcriptome sequences, putative TPS encoding genes were identified using BLASTX. Coding sequence of TPS1 was obtained from Ot young leaf cDNA using full-length ORF primers (Supplementary Table S1). TPS1 full-length ORF was cloned into the pRI 101-AN vector for over-expression in plants, using NdeI and BamHI restriction sites in forward and reverse primer (Supplementary Table S1), respectively. After verifying the construct by sequencing, it was transformed into the Agrobacterium tumefaciens (GV3101) chemically competent cells and plated on Luria-agar plate containing 50 µg/mL kanamycin and 25 µg/mL rifampicin. A. tumefaciens (GV3101) cells carrying over-expression construct were grown in 2 mL LB media with the above-mentioned antibiotics at 28 °C on rotary shaker set at 180 rpm for 2 days. This starter culture was used to inoculate 10 mL LB with the antibiotics and incubated overnight at 28 °C and 180 rpm. After incubation, cells were pelleted down by centrifugation at 10,000 rpm for 10 min at 4 °C. The cells were washed with half strength MS media, pH 5.4 and pelleted again. This cell pellet was re-suspended in half strength MS media, pH 5.4 to bring it to an O.D.600nm of 0.3–0.5. This suspension was used for syringe-assisted infection of the abaxial surface of the leaves. Three plants each were taken for over-expression, empty vector and uninfected control. A. tumefaciens (GV3101) cells harboring empty pRI 101-AN vector was used as control. The plants were kept at 22 °C, 18 h light and 6 h dark for acclimatization (3–4 days) before infection, and maintained at the same conditions for the entire course of the experiment. The samples for volatile and real-time analysis were collected after 4 and 8 days after inoculation (DAI), and were processed as described in the earlier sections.

Statistical analysis

GC–MS analyses of volatiles and gene expression analysis were performed with three biological and three technical replicates each. Data sets were represented as mean ± standard deviation. Significant differences between control and A. tumefaciens-treated plants for metabolites and gene expression were determined using two-way ANOVA followed by Bonferroni’s multiple comparisons test.

Results and discussion

Terpene diversity in different tissues across five Ocimum species

The terpene analysis indicated both quantitative (Fig. 1a, Supplementary Table S3) and qualitative (Fig. 1b, Table 1) differences in distribution of these compounds across different Ocimum species. We also analyzed the abundances of these metabolites in different tissues individually (Figs. 1c, 2a–e). Ten metabolites were common among different species, and were equally distributed between mono- and sesqui-terpenes. These ten common terpenes among Ocimum species could be represented as Ocimum specific chemical markers. In young leaves; linalool, ocimene, camphor and eucalyptol were the major monoterpenes in Og, Ok and Oa. Monoterpenes, ocimene and myrcene, were present in all the species studied. Young leaves were observed to be sesquiterpenes-rich in Og and Ot. Two species, Ok and Oa had comparable distribution of both mono- and sesqui-terpenes. Among sesquiterpenes, β-caryophyllene was detected in all the Ocimum species. A similar trend of metabolite distribution was observed in other tissues (Fig. 2a–e), where Og and Ot were found to be rich in sesquiterpenes, whereas in four different varieties of Ob species (Ob I–IV), monoterpenes dominated their metabolite profiles. An exception to this distribution was ocimene, which was found to be present in all the tissues of Og. This species-specific metabolite distribution was also evident from the cluster analysis, where species rich in either types of metabolite, clustered together. This chemical diversity can be attributed to the various ways that the intermediates can fold and cyclize in the reaction, catalyzed by different terpene synthases. The product of these reactions can be further modified by other enzymes such as P450 dependent mono-oxygenases and various transferases. In all the different tissues analyzed, monoterpene- rich Ob species and sesquiterpene-rich species Og and Ot clustered together.

Fig. 1.

Fig. 1

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Metabolite profiling of five species of Ocimum.a, b Venn diagram showing the unique and common metabolites from young leaves among five different species- a quantitative distribution and b qualitative distribution; c Heat map showing the distribution of top 10 metabolites from different Ocimum species in young leaves

Table 1.

Major terpenes present in different Ocimum species

graphic file with name 12298_2018_612_Tab1_HTML.jpg

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ND (not detected), OgI (Ocimum gratissimum I), OtI (Ocimum tenuiflorum I), Ok (Ocimum kilimandscharicum) and ObI (Ocimum basilicum I). Values are represented in relative area %. The results shown are an average of three biological replicates ± standard errors

Fig. 2.

Fig. 2

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Heat map showing relative abundance (%) of terpenoids from 10 Ocimum plants a Mature leaves, b Inflorescence, c Flower, d Stem and e Root. Different species and varieties are hierarchically clustered based on their abundance and diversity in mono- and sesquiterpenes

Transcriptome sequencing identifies 38 putative TPS transcripts in selected Ocimum species

Total RNA from leaf, inflorescence and stem tissue from O. gratissimum, O. tenuiflorum and O. kilimandscharicum were pooled and used for transcriptome analysis. With Illumina GA II platform, total of 40848829 (40.85 million) paired end reads (72 bp each), were created. In this, 38.25 million high quality reads (93.62%) were obtained with > 20 phred score and low-quality reads were trimmed. Velvet assembly generated a total of 220575 contigs with hash length of 55. These were used as input for Oases assembly to generate 253601 transcripts with average length of 255.9 bp. These transcripts were clustered to remove the redundancy using CD-HIT and generated 202081 unique transcripts. BLAST annotation of these transcripts was carried over with proteins of eudicotyledons family and the unannotated transcripts were further annotated with ESTs of Lamiids family. CAP3 analysis on these transcripts, gave 31034 contigs and 25855 singlets. Pathway annotation was performed by KAAS (KEGG Automatic Annotation Server). Out of the 56889 transcripts, only 4294 were assigned 1888 unique KO numbers. A web-based server Virtual Ribosome identified 20302 transcripts to have an open reading frame (ORF) more than 100 amino acids (aa), whereas 88 were without any ORF. Pfam analysis was carried out on the peptide sequence of the transcripts > 100aa, where Pfam ID was assigned to 15737 transcripts. In particular, we identified 38 putative TPSs, of which, 12 had similarities with monoterpene synthases, 13 were related to sesquiterpene synthases, 3 putative prenyl transferases; and 3 and 7 represented di- and triterpene synthases, respectively. In an attempt to understand the terpene accumulation in Ocimum species, three TPS contigs were selected and designated as TPS1 (Contig 19414), TPS2 (Contig 13517) and TPS3 (Contig 9641).

Three TPS shares sequence similarity with reported monoterpene synthases

Using amino acid sequence and protein function, TPSs have been organized into different sub-families, which includes, three angiosperm specific family (TPS-a, TPS-b and TPS-g), a gymnosperm specific subfamily (TPS-d), while TPS-c is most conserved among the land plants; TPS-e and f is conserved among the vascular plants (Bohlmann et al. 1998; Chen et al. 2011; Benabdelkader et al. 2015). Upon sequence analysis of these three contigs, presence of conserved domains and other characteristics of TPS gene family was evident (Fig. 3), including plastidial targeting sequence, characteristics of terpene synthases family TPS-b and TPS-g. TPS1 consists of 579 aa (63.7 kDa), TPS2 of 470 aa (51.7 kDa) and TPS3 of 580 aa (63.8 kDa), with TPS2 and 3 sharing 51% identity. TPS1 show 31% identity with TPS2 and 30% identity with TPS3. All three TPSs showed the presence of conserved features of plant TPSs like aspartate-rich region (DDxxD) which is involved in catalysis (Rynkiewicz et al. 2001) and xDx6E’ motif for metal cofactor binding (Degenhardt et al. 2009). A RR(X8)W motif was present in the N-terminal region of TPS2 and 3, which participates in the substrate ionization and is a characteristic of TPS-a and b subfamilies (Williams et al. 1998; Martin and Bohlmann 2004). Upon phylogenetic analysis, TPS2 and 3 grouped together with other monoterpene synthases belonging to TPS-b family of TPSs, which is in accordance with their identity score. TPS1 grouped together with linalool/nerolidol synthase from Solanum lycopersicum and nerol synthase from Glycine max, both of which belonged to Tps-g family of TPS (Fig. 4). This family includes mono- and sesqui-terpene synthase from angiosperms.

Fig. 3.

Fig. 3

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Sequence alignment of TPSs with other reported monoterpene synthases. Conserved regions are marked in red. ObFS- Ocimum basilicum Fenchol synthase, ObMyS- Ocimum basilicum Myrcene Synthase. TPS1, TPS2 and TPS3 from Ocimum species. RoCiS- Rosmarinus officinalis Cineole Synthase. Conserved regions are marked in red brackets and signal sequence indicated by red line

Fig. 4.

Fig. 4

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Phylogenetic analysis of TPS1, TPS2 and TPS3 (marked in blue box) with other reported TPS sequences from different classes (a–f). Neighbor joining tree was constructed using MEGA 6 with 1000 bootstrap iterations

Tissue specific expression analysis of three TPSs among different Ocimum species

Transcript levels of three TPSs was analyzed in leaves of five Ocimum species using semi-quantitative RT–PCR, and significant tissue and species-specific variations were observed. Across different tissues, in general, inflorescence represented highest expression levels of these contigs, while stem having the least. TPS1 transcript abundance was higher in leaf tissue of Ot, ObII and Oa, while TPS2 expression was higher in all the five species analyzed (Fig. 5a). In qRT–PCR, except for Ob species, TPS2 had very high expression levels in all tissues of the Ocimum species that were analyzed (Fig. 5b). In case of OtI and II, TPS1 and 2 had similar expression profiles. Apart from Ot, TPS1 was also found to be the most abundant contig in ObII, whereas in Og and Ok, it had low expression levels. On the other hand, TPS3 was observed to be abundant only in Ok, in both semi-quantitative and qRT–PCR.

Fig. 5.

Fig. 5

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Expression analysis of TPS1, TPS2 and TPS3 by a sqRT–PCR using 18S rRNA as reference gene in leaves tissue of five Ocimum species and b qRT–PCR analysis with EF1 as endogenous reference in different tissue of five species of Ocimum. Og- Ocimum gratissimum, Ot- Ocimum tenuiflorum, Ok- Ocimum kilimandscharicum, Ob- Ocimum basilicum, Oa- Ocimum americanum

Transient over- expression of TPS1 indicated to be a cis-β-terpineol synthase

In order to analyze gene function of TPS1, transient over-expression analysis was carried on Ok plants. Accumulation of metabolites and transcript upon over-expression of TPS1 was estimated by comparing them with that of control and empty vector infected plants (Fig. 5a, b). In qRT–PCR analysis, two-fold increase in TPS1 was noted, in both local and systemic tissues, which was reflected in 7% increase in accumulation of cis-β-terpineol in 8 DAI (Fig. 6 and Supplementary Table S2). There was no significant difference in accumulation of any other metabolites upon over-expression of TPS1 in Ok leaves. As compared to that of metabolite analysis, transcript abundance decreased at 8 DAI, an observation which was consistent with the reports suggesting instability of non-integrated T-DNA (Lacroix and Citovsky 2013).

Fig. 6.

Fig. 6

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acis-β-terpineol levels upon transient over-expression of TPS1 in O. kilimandscharicum. Experiment was performed with 3 biological and 3 technical replicates (L-local tissue, S- systemic tissue). Metabolite level was checked in leaf tissues collected after 4 and 8 days of infection. Data expressed in terms of relative area % and bars represents the SD values. bTPS1 expression levels determined by qRT–PCR. The values are expressed in comparison to the transcript levels in empty vector. Two-way ANOVA followed by Bonferroni’s multiple comparisons suggested significant difference between the data at p < 0.001 (indicated as ‘***’), p < 0.01 (indicated as ‘**’). c GC–chromatograms of metabolite analysis of control, empty vector infected and TPS1 infected plants. 1- cis-β-terpineol and 2- Linalyl acetate (internal standard). *Refers to the hydrocarbon dodecane observed in the GC–MS runs

Conclusion

Detailed terpene analysis of five different Ocimum species indicated significant variations in their distribution across different tissues and species. Transcriptome sequencing led to detection of 38 unique TPS transcripts. Sequence and phylogenetic analysis of three full-length putative TPS transcripts identified them as monoterpene synthases. Their transcript abundance in five tissues across Ocimum species revealed higher abundance of TPS2 in all the species, whereas TPS1 and TPS3 illustrated species-specific abundances. Further, Agro-infiltration based transient over-expression assay of TPS1 indicated it to be a cis-β-terpineol synthase.

Note The nucleotide sequences reported in this paper has been submitted to the GenBank with accession number MH925775 (TPS1), MH925776 (TPS2) and MH925777 (TPS3).

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 36 kb) (36.5KB, doc)
Supplementary material 2 (DOC 51 kb) (51KB, doc)
Supplementary material 3 (XLS 135 kb) (135KB, xls)

Acknowledgements

AA and SGL acknowledge Council of Scientific and Industrial Research (CSIR), New Delhi for SRF while SB acknowledges University Grants Commission, New Delhi, India for Kothari fellowship. The work was funded by CSIR-NCL-IGIB Joint Research program under XII Five Year Plan (BSC0124).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

References

  1. Anand A, Jayaramaiah RH, Beedkar SD, Singh PA, Joshi RS, Mulani FA, Dholakia BB, Punekar SA, Gade WN, Thulasiram HV, Giri AP. Comparative functional characterization of eugenol synthase from four different Ocimum species: implications on eugenol accumulation. Biochim Biophys Acta Proteins Proteomics. 2016;1864:1539–1547. doi: 10.1016/j.bbapap.2016.08.004. [DOI] [PubMed] [Google Scholar]
  2. Bach TJ. Some new aspects of isoprenoid biosynthesis in plants—a review. Lipids. 1995;30:191–202. doi: 10.1007/BF02537822. [DOI] [PubMed] [Google Scholar]
  3. Benabdelkader T, Guitton Y, Pasquier B, Magnard JL, Jullien F, Kameli A, Legendre L. Functional characterization of terpene synthases and chemotypic variation in three lavender species of section stoechas. Physiol Plant. 2015;153:43–57. doi: 10.1111/ppl.12241. [DOI] [PubMed] [Google Scholar]
  4. Bohlmann J, Crock J, Jetter R, Croteau R. Terpenoid based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)- α bisabolene synthase from grand fir (Abies grandis) Proc Natl Acad Sci USA. 1998;95:6756–6761. doi: 10.1073/pnas.95.12.6756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burke C, Croteau R. Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate. J Biol Chem. 2002;277:3141–3149. doi: 10.1074/jbc.M105900200. [DOI] [PubMed] [Google Scholar]
  6. Cane DE. Isoprenoid biosynthesis stereochemistry of the cyclization of allylic pyrophosphates. Acc Chem Res. 1985;18:220–226. doi: 10.1021/ar00115a005. [DOI] [Google Scholar]
  7. Cane DE. Enzymic formation of sesquiterpenes. Chem Rev. 1990;90:1089–1103. doi: 10.1021/cr00105a002. [DOI] [Google Scholar]
  8. Chen F, Tholl D, Bohlmann J, Pichersky E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011;66:212–229. doi: 10.1111/j.1365-313X.2011.04520.x. [DOI] [PubMed] [Google Scholar]
  9. Croteau R. Biosynthesis and catabolism of monoterpenoids. Chem Rev. 1987;87:929–954. doi: 10.1021/cr00081a004. [DOI] [Google Scholar]
  10. Davis EM, Croteau R. Cyclization enzymes in the biosynthesis of monoterpenes, sesquiterpenes and diterpenes. New York: Springer; 2000. pp. 53–95. [Google Scholar]
  11. Degenhardt J, Koellner TG, Gershenzon J. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry. 2009;70:1621–1637. doi: 10.1016/j.phytochem.2009.07.030. [DOI] [PubMed] [Google Scholar]
  12. Dixon RA. Plant natural products: the molecular genetic basis of biosynthetic pathway. Curr Opin Biotech. 1999;10:192–197. doi: 10.1016/S0958-1669(99)80034-2. [DOI] [PubMed] [Google Scholar]
  13. Eisenreich W, Schwarz M, Cartayrade A, Arigoni D, Zenk MH, Bacher A. The deoxyxylulose phosphate pathway of terpenoid biosynthesis in plants and microorganisms. Chem Biol. 1998;5:R221–R233. doi: 10.1016/S1074-5521(98)90002-3. [DOI] [PubMed] [Google Scholar]
  14. Garcia R, Alves ESS, Santos MP, Aquije GMFV, Fernandes AAR, Santos RB, Ventura JA, Fernandes PMB. Antimicrobial activity and potential use of monoterpenes as tropical fruits preservatives. Braz J Microbiol. 2008;39:163–168. doi: 10.1590/S1517-83822008000100032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hampel D, Mosandl A, Wust M. Biosynthesis of mono- and sesquiterpenes in carrot roots and leaves (Daucus carota L.): metabolic cross talk of cytosolic mevalonate and plastidial methylerythritol phosphate pathways. Phytochemistry. 2005;66:305–311. doi: 10.1016/j.phytochem.2004.12.010. [DOI] [PubMed] [Google Scholar]
  16. Jennewein S, Croteau R. Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biot. 2001;57:13–19. doi: 10.1007/s002530100757. [DOI] [PubMed] [Google Scholar]
  17. Krithika R, Srivastava PL, Rani B, Kolet SP, Chopade M, Soniya M, Thulasiram HV. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis. Sci Rep. 2015;5:8258. doi: 10.1038/srep08258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lacroix B, Citovsky V. The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation. Int J Dev Biol. 2013;57:467–481. doi: 10.1387/ijdb.130199bl. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lesburg CA, Caruthers JM, Paschall CM, Christianson DW. Managing and manipulating carbocations in biology: terpenoid cyclase structure and mechanism. Curr Opin Struc Biol. 1998;8:695–703. doi: 10.1016/S0959-440X(98)80088-2. [DOI] [PubMed] [Google Scholar]
  20. Martin DM, Bohlmann J. Identification of Vitis vinifera (−)-α-terpineol synthase by in silico screening of full-length cDNA ESTs and functional characterization of recombinant terpene synthase. Phytochemistry. 2004;65:1223–1229. doi: 10.1016/j.phytochem.2004.03.018. [DOI] [PubMed] [Google Scholar]
  21. McGarvey DJ, Croteau R. Terpenoid metabolism. Plant Cell. 1995;7:1015–1026. doi: 10.1105/tpc.7.7.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Poulter CD. Farnesyl diphosphate synthase. A paradigm for understanding structure and function relationships in E-polyprenyl diphosphate synthases. Phytochem Rev. 2006;5:17–26. doi: 10.1007/s11101-005-4887-1. [DOI] [Google Scholar]
  23. Ramesha HJ, Anand A, Beedkar SD, Dholakia BB, Punekar SA, Kalunke RM, Gade WN, Thulasiram HV, Giri AP. Functional characterization and transient expression manipulation of a new sesquiterpene synthase involved in β-caryophyllene accumulation in Ocimum. Biochem Biophys Res Commun. 2016;473:265–271. doi: 10.1016/j.bbrc.2016.03.090. [DOI] [PubMed] [Google Scholar]
  24. Rodriguez-Concepion M. The MEP pathway: a new target for the development of herbicides, antibiotics and antimalarial drugs. Curr Pharm Des. 2004;10:2391–2400. doi: 10.2174/1381612043384006. [DOI] [PubMed] [Google Scholar]
  25. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J. 1993;295:517–524. doi: 10.1042/bj2950517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rynkiewicz MJ, Cane DE, Christianson DW. Structure of trichodiene synthase from Fusarium sporotrichioides provides mechanistic inferences on the terpene cyclization cascade. Proc Natl Acad Sci USA. 2001;98:13543–13548. doi: 10.1073/pnas.231313098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sprenger GA, Schorken U, Wiegert T, Grolle S, de Graaf AA, Taylor SV, Begley TP, Bringer-Meyer S, Sahm H. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc Natl Acad Sci USA. 1997;94:12857–12862. doi: 10.1073/pnas.94.24.12857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–2729. doi: 10.1093/molbev/mst197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tantillo DJ. Biosynthesis via carbocations: theoretical studies on terpene formation. Nat Prod Rep. 2011;28:1035–1053. doi: 10.1039/c1np00006c. [DOI] [PubMed] [Google Scholar]
  30. Thiel R, Adam KP. An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants. Phytochemistry. 2002;59:269–274. doi: 10.1016/S0031-9422(01)00453-8. [DOI] [PubMed] [Google Scholar]
  31. Thulasiram HV, Poulter CD. Farnesyl diphosphate synthase: the art of compromise between substrate selectivity and stereoselectivity. J Am Chem Soc. 2006;128:15819–15823. doi: 10.1021/ja065573b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams DC, McGarvey DJ, Katahira EJ, Croteau R. Truncation of limonene synthase preprotein provides a fully active ‘pseudomature’ form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair. Biochemistry. 1998;37:12213–12220. doi: 10.1021/bi980854k. [DOI] [PubMed] [Google Scholar]

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