© NIAB 2016 Plant Genetic Resources; 1–14

ISSN 1479-2621 doi:10.1017/S1479262116000162

Medicinal plant transcriptomes: the new

gateways for accelerated understanding
of plant secondary metabolism
Sandhya Tripathi, Jyoti Singh Jadaun, Muktesh Chandra and Neelam S. Sangwan*
Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants
(CSIR-CIMAP), Lucknow-226015, UP, India

Received 11 April 2016; Accepted 11 April 2016

Abstract
Medicinal plants are the vital source of numerous structurally diverse pharmacologically active me-
tabolites collectively called as secondary metabolites finding extensive applications in traditional
systems of medicine and in pharmaceutical industries. Several distinctive and complex pathways
operate in an interactive manner via metabolic networks that are responsible for the accumulation
of such highly specialized metabolites. Secondary metabolites are believed to play a wide spectrum
of physiological and functional roles in plants, many of which being investigated and supported by
the experimental studies. Biosynthetic pathway related studies on various aspects in these medicinal
plants have been found very tedious owing to several issues in plants such as considerably lower
metabolite concentrations in native tissues, existence at different locations, and highly complex
multi-step pathways, etc. Pathway elucidation and gene/enzyme discovery for studying metabolic
pathway evolution and subsequent engineering could be better achieved by mining various path-
way databases and reconstruction of metabolic networks available at different omics databases.
Though medicinal plants have a limited range of genomic sequences available, however recently,
next generation sequencing is being widely used to generate a comprehensive transcriptomic re-
source for these plants. It is anticipated that databases and resources generated from these studies
are likely to play a key role towards the study and exploitation of metabolites from medicinal plants
in near future. In this review, we have discussed next generation sequencing approaches, which
were used for the generation of transcriptomic resources for several medicinally important plants.
The relevance of transcriptomic approaches in curation of the pathways linked with the synthesis of
major secondary metabolites along with their precursors of pharmaceutical importance in medicinal
plants is also comprehensively analysed.

Keywords: ashwagandha, NGS, essential oils, plant secondary metabolism, terpenoids,

transcriptome

Introduction of data of considerable importance. Among these the de

The past decade has witnessed an upsurge in the knowl-
edge of genomic and transcriptomic resources. Biologists
are now utilizing next generation sequencing (NGS)
technologies for generation and analysis of vast amounts
*Corresponding author. E-mail: nss.cimap@gmail.com
novo transcriptome sequencing has become one of the
most sought after tool for an easy resource to gain informa-
tion (Oksman-Caldentey et al., 2004; Sangwan et al., 2013,
2014). Steady decline in the cost of sequencing has facili-
tated a cost and labour effective data flow through this tech-
nology (Egan et al., 2012, Xiao et al., 2013). Transcriptome
sequencing is preferred as it provides vital information by
being influenced by different developmental stages and
several physiological factors like environmental changes,
2 S. Tripathi et al.
etc. unlike that in genome. Therefore, the technology is
supporting the possibility of generation of comprehensive
view of various pathways involved in the secondary metab-
olism of medicinal and aromatic plants. (Gupta et al., 2013;
Sangwan et al., 2013).
Medicinal plants transcriptomes: an emerging
concept
Plants are able to produce an array of organic molecules.
Based on their utility for plant survival; these compounds
can be categorized into two classes viz primary and sec-
ondary metabolites (Singh-Sangwan et al., 1994).
Secondary metabolites comprise enormously distributed
group of chemicals synthesized in various kingdoms of
life like plants more often and additonally fungi, algae, bac-
teria and animals. Previously, these compounds were
called as secondary because their absence or any alteration
in their level in plants does not lead to lethal effects unlike
primary metabolites, which are essential for growth and de-
velopment. Recently, this old concept about the secondary
metabolites has been revised and it is largely believed and
proven in increasing cases that secondary metabolites play
a very important role in plant development, adaptation and
defence (Bose et al., 2013; Yadav et al., 2014; Nick, 2015). A
lot of studies have been conducted to reveal the functional
significance of secondary metabolites in medicinal plants,
the information available in this respect is however avail-
able in scattered manner (Chowhan et al., 2013; Lee
et al., 2015; Sunohara et al., 2015).
Medicinal plants were being used traditionally since time
immemorial throughout the world including India and
China, being the major producers. These plants form the
basis of origin and development of herbal formulations
(Sangwan et al., 2003). In some plants like Withania som-
nifera, Azadirachita indica, Mentha species, all parts have
the medicinal properties, but in others these properties are
tissues specific and even cell specific like production of
vincristine and vinblastine is reported only in leaves
(Kushwaha et al., 2013; Narnoliya et al., 2014; Sangwan
et al., 2014; Pan et al., 2016). Fragrances of oils extracted
from aromatic plants such as Geranium, Cymopogan,
Ocimum species and Eucalyptus have medicinal proper-
ties. This is the reason why these are used in aromatherapy.
The therapy was previously used in some countries of the
world but recently it has evolved as a modern therapy to
relieve the tension and depression resulting in mental re-
laxation (Cooke and Ernst, 2000; Fakari et al., 2015).
These essential oils are also used as a constituent in formu-
lations of many cosmetic products (Sangwan et al., 2003;
Wang et al., 2016). Resurgence in plant-based herbal medi-
cine is due to their fewer or none side effects unlike the
allopathic and synthetic medicines. These compounds
are synthesized via various complex and interconnected
metabolic processes and limited information is available
about their biosynthetic pathways. So studies on biosyn-
thetic pathways related to genetic mechanism for the syn-
thesis of these compounds are in demand (Kushwaha et al.,
2013; Srivastava et al., 2015; Mishra et al., 2016). Secondary
metabolites such as withanolides, artemisinin, terpenes,
vincristine and vinblastine are synthesized by different
plants through different pathways such as alkaloid, phenl-
propanoid, terpenoid pathway, etc. (Sangwan et al., 2003,
2015; Sangwan and Sangwan, 2014; Jadaun et al., 2016;
Mishra et al., 2016). The transcriptomes of many important
medicinal plants and their different parts as well such as
leaf, root and stem, etc. have been reported so far (Gupta
et al., 2013; Narnoliya et al., 2014; Rajakani et al., 2014;
Rastogi et al., 2014). Elucidation of pathway for the biosyn-
thesis of secondary metabolites via different genes through
transcriptomic approaches have been attempted in several
cases. This will open up the possibility to uncover novel
gene families with discrete functions using newer set of
plant specific transcripts (Kumar et al., 2012; Yamazaki
et al., 2013; Rastogi et al., 2014). An account of the medicin-
al plant transcriptomes published till date with the details
such as species and the strategies used to sequence the par-
ticular tissue of the plant shows a sharp trend of the devel-
opment in the area (Table 1).
Transcriptomes of some of the most important plants
such as Salvia miltiorrhiza, Artemisia annua, Withania
somnifera, Centella asiatica, Catharanthus roseus and
Ocimum species, etc. are presently available (Wenping
et al., 2011; Sangwan et al., 2013; Soetaert et al., 2013; De
Bernonville et al., 2015; Rastogi et al., 2014; Gupta et al.,
2015). Recently, our group had reported the de novo se-
quencing of the leaf transcriptome of Centella asiatica,
which was one of earliest such attempt for medicinal plants
in Apiaceae family (Sangwan et al., 2013). Another major
leap of progress in de novo transcriptomes from medicinal
plant is generation of transcriptome data for Indian medi-
cinal plant neem (Krishnan et al., 2012). Combined report
of comparative account of genome and four transcriptomes
for neem (Azadiracta indica) reveals many key steps in-
volved in neem secondary metabolic pathway (Krishnan
et al., 2012). Neem is the first member of Meliaceae family
to be sequenced, analysed for phylogentic study and com-
pared with other higher order plants in terms of sequence
similarity. Our group at CSIR-CIMAP, Lucknow further ana-
lysed the tissue specific expressed sequenced tags (ESTs)
generated in suppression subtractive hybridization ap-
proaches to further add to the knowledge of important
pathway transcripts (Narnoliya et al., 2014). The approach
not only gave highly tissue specific transcripts but also
helped us to report first full length gene from neem,
which is hydroxyl methyl glutaryl CoA enzyme A reductase
(HMGR) a key regulatory gene in the biosynthesis of
Medicinal plant transcriptomes 3

Table 1. Transcriptomic resources from medicinal plants

Secondary metabolic
Plant Tissue Approach pathway References

Azadirachta indica Root, leaf, stem and Illumina TruSeq™ library Terpenoid pathways Krishnan et al.
flower (2012)
Azadirachta indica Fruit mesocarp and Ssuppression subtractive Limonoid pathway Narnoliya et al.
endocarp hybridization (2014)
Azadirachta indica Fruit and leaf Suppression subtractive Limonoid pathway Rajakani et al.
hybridization (2014)
Asparagus Leaf and root Illumina Hi-seq GAII Analyzer/ Steroidal sapogenein Upadhyay et al.
racemosus 454 GS FLX/5500 SOLiD biosynthesis (2014)
System.
Bupleurum Root 454 pyrosequencing Saikosaponin biosynthesis Sui et al. (2011)
chinense
Costus pictus Leaf Illumina GAIIx Bixin biosynthesis Annadurai et al.
(2012)
Catharanthus Shoots Illumina HiSeq2000 RNA Alkaloid biosynthesis Moerkercke
roseus sequencing et al. (2013)
Centella asiatica Leaf Illumina Genome analyzer II Triterpenoids Sangwan et al.
Platform (2013)
Camptotheca Leaf 454 GS FLX platform shotgun Camptothecin Sun et al. (2012)
acuminate sequencing biosynthesis
Panax notoginseng Root 454 pyrosequencing technology Triterpenoid saponins Luo et al. (2011)
Panax ginseng Roots, stems, leaves 454 pyrosequencing Ginsenoside biosynthesis Li et al. (2013)
and flowers
O. sanctum and Leaf Illumina HiSeq1000 platform Phenyl propanoid and Rastogi et al.
O. Basilicum terpenoid pathway (2014)
Ipomoea batatas Root Illumina paired-end sequencing Brasinosteroid pathway Wang et al.
(2010)
Boehmeria nivea Leaf, root, stem, HiSeqTM 2000 platform Fiber biosynthesis Liu et al. 2013
stem xylem and (Illumina)
stem
Cicer arietinum Root and shoot Illumina Genome Analyzer II Garg et al.
platform (2011)
Hevea brasiliensis Leaf and Latex Illumina sequencing Rubber (Isoprenes) Xia et al. (2011)
biosynthesis
Epimedium Young leaves 454 GS-FLX pyrosequencing Flavonoid pathway Zeng et al.
sagittatum (2010)
Hippophae Leaf Illumina Solexa GA II platforms Cold stress Sharma et al
rhamnoides 2012
Salvia miltiorrhiza Leaf Solexa deep sequencing Phenylpropanoid and Wenping et al.
terpenoid pathways (2011)
Withania somnifera In vitro cultured leaf Illumina Hiseq2000 Withanolide biosynthesis Senthil et al.
and root 2015
Withania somnifera Leaf and root 454-GS FLX sequencing platform Withanolide biosynthesis Gupta et al.
(2013)

several isoprenoids including limonoids in neem (Rajakani
et al., 2014; Narnoliya et al., 2014). Also various lipids and
storage protein specific transcripts were located and as-
signed to mesocarp, endocarop, leaf and whole fruit
(Narnoliya et al., 2014). Further, specific gene families
such as glucosyltransferase and cytochrome P450 plays a
major role in isoprenoid diversification attributing specifici-
ties to the chemical moieties as well as to the native produ-
cer system. The information gained from the transcriptomic
data could be retrieved and further validated using wet
4 S. Tripathi et al.
laboratory experiments, as in case of Neem deeper insights
into the molecular biology in a much shorter time span
have been achieved, which was difficult using traditional
approaches. In case of Azadirichta indica, attempts have
located specific cytochrome P450, which are putative can-
didates involved in neem isoprenoid pathway (Rajakani
et al., 2014). Despite the presence of enormous informa-
tion on chemical aspects in Neem, no omic studies have
been reported so far and it constitutes the first ever reports
on in silico studies in Neem secondary metabolism. The in
depth studies related to transcriptome in terms of its
assembly, annotation and profiling of transcripts for path-
way genes for secondary metabolism with their expres-
sion analysis would establish a milestone to improve the
plants in study and related members using genetic engin-
eering and molecular marker studies. (Sangwan et al.,
2013; Mishra et al., 2015; Mishra et al., 2016; Jadaun et al.,
2016).
It is believed that the plants are being protected from in-
sect predation by the natural plant products; many of which
are synthesized and accumulated in the glandular trichomes
(Wagner et al., 2004; Sangwan et al., 2014). Studies have
shown that localized secondary metabolites such as essen-
tial oil is being stored in capitate and peltate trichomes
(Sharma et al., 2003; Bose et al., 2013; Odigmegwu, 2013;
Yadav et al., 2014). A. annua is an important medicinal
and aromatic plant synthesizing monoterpene oil and sev-
eral non-volatile sesquiterpenes including anti-malarial arte-
misinin, which was recognized for awarding the 2015 Nobel
Prize in Physiology or Medicine (McKerrow, 2015). The
availability of contigs related to biosynthesis of terpenoids
and flavonoids predicts their important role in metabolism
of glandular trichomes. A detailed study in relation to
genes of trichome would help in discovery of novel genes
in addition to illuminating the mechanism of secondary me-
tabolism regulation and functioning of trichomes (Bose
et al., 2013; Yadav et al., 2014).
Regardless of huge importance of secondary metabolites
in human and plant life, the availability of genomic informa-
tion in terms of plant secondary metabolism is still limited es-
pecially in case of medicinal plants, where no model plant
have been characterized yet. Transcript profiling of jasmo-
nate elicited tobacco based on cDNA-amplified fragment
length polymorphism was carried out in combination with
the functional genomics-based approach, provided a reper-
toire of novel genes for plant secondary metabolite analysis.
The results presented a remarkable reprogramming at genet-
ic level affecting the metabolism in response to jasmonic acid
and were well in confirmation to the shifts observed in the
biosynthesis of the investigated metabolites. The gene tags
and the transcriptomic data obtained as above will serve as
the basis for detailed metabolic engineering of medicinal
plants in particular through creation of novel tools.
Moreover, the comparative studies in this respect
complement the specificity along with the significant fea-
tures of cDNA-AFLP transcript profiling for unravelling
plant secondary metabolism in the absence of model system.
The issues pertaining to the biosynthesis and chemoty-
pic diversity related to the existence of commercially im-
portant secondary metabolites such as withanolides have
been the subject of study in recent past (Chaurasiya et al.,
2009). Comparison of both root and leaf leads to the iden-
tification of many unigenes involved in the secondary me-
tabolism pathways; all these studies are being done by the
homology. The informative knowledgebase is thus estab-
lished for Withania and it will unravel tissue specific path-
way mechanisms in addition to the development of
advanced strategies to increase the withanolide biosyn-
thesis using modern approaches of biotechnology
(Sangwan et al., 2014, 2015). The linking of gene expres-
sion data to genetic information is also of great importance
to understand the interplay among genome, transcriptome
and phenotypic expression. The current trend and tool de-
velopment in the area of biological sciences therefore ne-
cessitate the demand of high throughput transcriptome
sequencing for generation of large-scale transcriptomic re-
sources important for discovery of novel genes and devel-
opment of molecular markers (Sangwan et al., 2014).
The present scenario of maturation and development of
new sequencing technologies day by day had set the re-
quirement of revision in the improvement of existing com-
putational tools (Table 2). Allelic variation and RNA
processing events are the other factors, which should be
taken into consideration while development of newer re-
construction methods for utilizing the information retained
by the expressed transcript sequences generated through
RNA-Seq (Garber et al., 2011). Moreover, metatranscrip-
tomics is also an emerging area as a consequence of ad-
vancements in de novo transcriptome assembly. In this
discipline, simultaneous investigation of thousands of tran-
scriptome from the whole microbial community is per-
formed. Constant improvement in RNA-Seq experiments
and related protocols will provide regular solutions to the in-
formatics challenges.
NGS approaches to study transcriptomes
Study of transcriptome through de novo sequencing and
further assembly is significantly accelerated since it was
published first by generation of pass reads of cDNA clones
as ESTs in 1993 (Adams et al., 1993). The study of transcrip-
tomes has been tremendously advanced by the develop-
ment of NGS technologies along with the novel
complementary tools at a common platform. This in turn fa-
cilitates the inexpensive generation of billions of parallel
reads ranging from 50 to 800 bp based on features charac-
teristic to different technologies. These majorly include
Medicinal plant transcriptomes 5

Table 2. Description of various tools and platforms for NGS analysis

Supported
Tool Role Description Platform OS Reference

Trinity Assembly Requires paired reads Illumina Linux Grabherr et al. (2011)
Trans-ABySS Assembly Addresses variation in Illumina Linux/Mac Robertson et al. (2010)
local read densities OS X
SOAPdenovo Assembly Construct full-length Illumina Linux Luo et al. (2012)
transcript sets
Velvet Assembly Requires paired reads 454, Illumina Linux Zerbino and Birney (2008)
Rnnotator Assembly Reconstruct full-length Illumina Linux Martin et al. (2010)
genes
Oases (depends on Assembly Clusters the contigs into llumina, Linux http://www.ebi.ac.uk/~zerbino/
Velvet) loci SOLiD, or oases/
454
MIRA Assembly Performs true hybrid 454, Illumina Linux Chevreux et al. (2004)
assemblies
CAP3 Assembly clustering reads 454 Linux Huang and Madan (1999)
gsAssembler Assembly Uses Newbler; 454 Linux Roche/454 Life Sciences,
GUI-based Branford, Connecticut, USA

technologies like Roche/454 (Margulies et al., 2005),
Illumina (Bentley et al., 2008) and AB SOLiD (McKernan
et al., 2009). However, the recent upsurge of a new era
of next i.e. third-generation sequencing has helped the pro-
duction of sequences with greater read lengths as com-
pared with previous ones having lower per run cost and
in a much less time. Further, the decreasing cost of sequen-
cing and rise in data output in addition to the sample
throughput for the second- and third-generation sequen-
cing technologies have opened up plant transcriptomics,
genomics and breeding community, to gather novel infor-
mation in turn. RNA sequencing is therefore, replacing
other methods for gene expression related studies like ser-
ial analysis of gene expression and microarrays. It provides
information about expressed sequences present at a specif-
ic point of time in a particular tissue due to its feature of se-
quencing to a greater depth to mine out the rare transcripts
otherwise not detectable. Thus, it provides a clearer picture
of events occurring in a tissue transcriptome for example
information linked with novel transcripts (Denoeud et al.,
2008), gene expression (Sangwan et al., 2013), single nu-
cleotide polymorphisms (SNPs) (Alagna et al., 2009), alter-
native splicing (Wang et al., 2008), structure variation
(Maher et al., 2009) and more importantly gene characteriza-
tion (Alagna et al., 2009; Wang et al., 2009; Brautigam et al.,
2011).
Since now no model species in case of medicinal plants
have been defined and thus the next generation sequen-
cing more commonly called as RNA-Seq is playing even
greater role in unravelling the transcriptomic events occur-
ring in a specific tissue at a specific situation (Fig. 1).
However, for medicinal plants limited information is avail-
able with respect to the genome in terms of ploidy, size,
heterozygosity and content of repetitive sequences.
Additionally, research in medicinal plants is still restricted
to biochemical pathways and downstream pharmacologic-
al properties in animal and human systems. Therefore, RNA
seq is proved to be a practical tool for studying non-model
species, in which very little or no genetic information is
available as it focuses on sequencing of the coding regions
instead of the whole genome. Many medicinal and aromat-
ic plants have recently being sequenced by these ap-
proaches such as Eleutherococcus senticosus (Hwang
et al., 2015) Bupleurum sp (Sui et al., 2015), Uncaria
rhynchophylla (Guo et al., 2014) Stevia rebaudiana
(Chen et al., 2014) Rhazya stricta (Park et al., 2014) C. asia-
tica (Sangwan et al., 2013), Azadirachta indica (Krishnan
et al., 2012), W. somnifera (Gupta et al., 2013), Aquilaria
sinensis (Wu et al., 2013), Panax quinquefolius (Sun et al.,
2010), Euphorbia fischeriana (Barrero et al., 2011; Luo
et al., 2011), Panax ginseng C. A. Meyer (Li et al., 2013)
A. annua (Wang et al., 2009) and olive (Alagna et al.,
2009), etc.
Experimental design for NGS sequencing
The basic planning and methodology for RNA – Seq experi-
mental design involves, sample selection initially, in which
information about the species to be sequenced is taken into
consideration in addition to the information related with tis-
sue developmental stages, treatment type, etc. For optimized
6 S. Tripathi et al.
Fig. 1. Transcriptomic workflow overview.
assembly and data interpretation, single established che-
motype/genotype must be the source of RNA isolation
(Fig. 1). Further, the background information of the plant
species is also important in terms of its homozygous and
heterozygous nature. Duplication events resulting in diver-
gence of a species from other closely related species must
be considered.
The major prerequisite for RNA sequencing is the selec-
tion of a suitable platform. Currently two important NGS
technologies are used successfully for studying non-model
plants, Roche/454 (Margulies et al., 2005) and Solexa/
Illumina (San Diego, California, USA). Some new technolo-
gies such as Ion torrent (Life Technologies, Carlsbad, CA,
USA), Pac Bio (Pacific Biosciences, Menlo Park, CA, USA)
and Helicos (Helicos Biosciences, Cambridge, MA, USA)
are also emerging, which promise long-read length and
high-quality data eliminating the difficulty to assemble
the data. After sequencing the raw reads, the reads must
be checked for quality, contamination and curation.
Assembly process based on mapping to a reference se-
quence or de novo method is then performed on cleaned
and filtered reads. Several assemblers are presently avail-
able, which assemble short reads generated by NGS tech-
nologies based on different alogorithms and different
parameters. The most popular assemblers for the assembly
of short reads generated through NGS includes
gsAssembler (454 Life Sciences), Mira (Chevreux et al.,
2004), Velvet (Collins et al., 2008), Trinity (Grabherr
et al., 2011), etc.
The ultimate step involves the characterization and func-
tional annotation of the transcriptome, which involves de-
piction of gene repertoire in the organism by comparative
analysis. Annotation is generally done using BLAST, which
retrieves significant hits with previously annotated genes in
the non-redundant database. Combined annotations for
this purpose could be best provided by available tools
like Blast2GO (Conesa et al., 2005; Conesa and Götz,
2008; Götz et al., 2008), etc. The information thus obtained
combined with more information related to metabolic path-
ways retrieved from Kyoto Encyclopedia of Genes and
Genomes (KEGG) or similar databases could be of great
importance in understanding the underlying mechanism
at genetic or molecular level (Götz et al., 2008). Further
downstream analysis may also include SNP analysis, in
which single base pair variation is detected having its appli-
cation in generation of genetic and molecular markers,
which greatly focus on expressed regions having higher
likelihood of phenotypic effect. The detection involves
the usage of different techniques for reference – guided as-
sembly or de novo assembly.
Moreover, expression analysis may also be performed by
monitoring the changes in gene expression in different tis-
sues, spatial or temporal, or in response to different treat-
ments to understand the pattern of genes critical in
respective conditions. The tools may vary based on avail-
ability of genome reference sequence (Li and Durbin,
2009).
Transcriptomics in relation to secondary
metabolic pathway
Medicinal plants are rich in metabolites and some are con-
siderably recalcitrant to handle for molecular analysis. This
type of analysis could be implemented through whole gen-
ome sequencing or through transcriptomics approach
(Sangwan et al., 2013, 2014). Presently, transcriptomics ap-
proach through RNA-seq technique has received more at-
tention rather than whole genome sequencing due to its
rapid data outputs, less complexity, less time consumption
in assembly and cost effectiveness. Another reason for
shifting towards RNA-seq technique could be the huge gen-
ome size, various ploidy levels. Plants have approximately
20,000–60,000 genes and only 15–25% of these genes con-
tributed towards the pathways leading to secondary meta-
bolites production (Góngora-Castillo et al., 2012).
Previously, microarray was used to detect the expression
pattern of genes in two types of organism, tissue or in
same organism at different time period but now RNA-seq
has overcome microarray technique. In RNA-seq expres-
sion of thousands genes can be monitored simultaneously
Medicinal plant transcriptomes
and there is no need of previous information about the gen-
ome to make probes as in microarray. Day by day enrich-
ment is taking place in sequencing technology such as
recent advancement from Sanger’s method to high
throughput sequencing platforms like Roche pyrosequen-
cing 454 (Margulies et al., 2005), Illumina (Bentley et al.,
2008) and SoLiD™ (McKernan et al., 2009) and data ana-
lysis tools enables us to decode the metabolic complexity
of medicinal plant at genetic level to some extent. In case
of medicinal plants Roche pyrosequencing and IIIumina
high throughput sequencing are most commonly used plat-
forms for generation of transcriptomic data of several im-
portant medicinal plants (Hao et al., 2012; Sangwan
et al., 2013, Yang et al., 2013; Bose and Chattopadhyay,
2016; Deng et al., 2016; Jain et al., 2016). Medicinal plants
have a very huge resource for bioactive molecules, which
can act as a template molecule for drug designing and
model development against many incurable diseases.
There is a huge potential of discovering more new drugs
from plant-based sources (Mondal et al., 2012). So, it is of
prime requirement for researchers to have a system com-
prising the information about all the medicinal plants in
an organized manner, which can be further used for bene-
fits of human beings. Major part of bioactive molecules de-
rived from plants is under the category of secondary
metabolites. So secondary metabolites consist of a plethora
of compounds having diversity in their synthesis as well as
in chemical skeleton, so for convenience to understand
their origin, biosynthesis and functional aspects, these
compounds are broadly classified in three groups namely
terpenoid, alkaloid and phenyl-propanoids.
Elucidation of terpenoid pathway through
transcriptomic approach
Terpenes constitute the largest group of plant natural pro-
ducts and building block of terpene is the five carbon com-
pound, isoprene, so these are also called isoprenoids.
Number of monomers vary from 5 (hemiterpene) to 1000
(rubber) and in structure it may be linear or polycyclic.
On the basis of carbon number present in molecule these
can be classified in many classes such as hemiterpene con-
taining 5 carbon atoms (C5), monoterpene containing 10
carbon atoms (C10), sesquiterpene containing 15 carbon
atoms (C15), diterpene containing 20 carbon atoms
(C20), triterpne containing 30 carbon atoms (C30), tetrater-
pene containing 40 carbon atoms (C40, eight isoprene
unit), polyterpene (more than eight isoprene unit).
Terpenes assist in defence mechanism of plant through
emission of volatiles. These also constitute a large group
of pharmaceutically important chemicals, which are used
by humans since ancient time for example artemisinin
from A. annua was used as an antimalarial drug, use of
7
Mentha species for menthol production, withanolides
from W. somnifera, asiaticosides from C. asiatica
(Sangwan et al., 2010; Chaurasiya et al., 2009; Singh
et al., 2014). There are many evidences for the occurrences
of two pathways for terpene biosynthesis; one is cytosolic
mevalonate (MVA) and another is plastidial non-
mevalonate or methyl erythritol phosphate (MEP) pathway
(Sangwan et al., 2008). But very scanty literature is avail-
able about the biosynthesis mechanism and also for genetic
regulation of terpenoid biosynthesis. So there is a great ne-
cessity to generate more knowledge about the biosynthetic
pathways and this problem has been solved up to a large
extent through outbreak of NGS approaches. There are
several examples of medicinal plants, in which transcrip-
tomics approach is followed to elucidate the terpenoids
metabolism such as Ilex asprella (Zheng et al., 2014) C.
asiatica (Sangwan et al., 2013), W. somnifera (Gupta
et al., 2013), Panax notoginseng (Luo et al., 2012), S. mil-
tiorrhiza (Yang et al., 2013), Costus pictus (Annadurai
et al., 2012). In case of C. asiatica transcriptome,
Sangwan et al. (2013) reported that KEGG analysis reveals
the presence of enzymes related to both biosynthetic path-
ways (MVA and MEP) of terpenes and key genes of terpen-
oid metabolism such as limonene synthase, of
monoterpenoids, farnesyl diphosphate synthase (FPPS)
for sesquiterpenoids and, β-amyrin synthases (BAS), cy-
cloartenol synthases (CAS), squalene epoxidase (SQE),
squalene synthases (SS), etc. associated with triterpenoid/
steroids were present in abundance. Similarly in W. somni-
fera GO and KEGG analysis results showed the full map-
ping of genes encoding enzymes involved in withanolide
biosynthesis (Gupta et al., 2013). Other examples of
usage of transcriptome analysis for identification of gene
related terpenoid pathways like saikosaponins biosyn-
thesis in Bupleurum chinense (Sui et al., 2011) tanshinone
biosynthesis in S. miltiorrhiza (Yang et al., 2013), ginko-
sides biosynthesis in P. ginsing (Chen et al., 2011), taxol,
a most potent anticancer drug, biosynthesis in Taxsus cap-
sidata (Wu et al., 2011). Artemisinin, the extensively stud-
ied potent antimalarial drug from A. annua is
sesquiterpene lactone produced in the glandular trichomes
of the plant. Transcriptome of glandular trichome of A.
annua was reported using 454 pyrosequencing. Several
relevant genes and transcripts related with secondary meta-
bolic pathway specially artemisnin were annotated and
their association with the biosynthesis and regulation of ar-
temisnin was revealed (Wang et al., 2009). Comparison of
described pathway genes with trichome ESTs collections
from other plants have shown that a common pattern of
genes were expressed, which were assigned to similar
functional categories in different plant species. This was
also confirmed by quantitative polymerase chain reaction
analysis using selected unigenes and novel transcripts in
the plant. Saponins possess biological activities also aid
8 S. Tripathi et al.
in human health, de novo assembly of leaf and root tissues
provides a close view of triterpene related pathway genes
in Asparagus racemosus (Upadhyay et al., 2014).
Elucidation of alkaloid pathway through
transcriptomics approach
Alkaloids are a group of heterocyclic nitrogen containing
compounds, which are bitter in taste. Approximately
10,000 alkaloids are reported from different sources, alka-
loids are not only restricted to plant kingdom but its occur-
rence have been reported in animals also. Alkaloids protect
the plants against herbivore and pathogen attack. The mo-
lecular weight of alkaloid varies from simple amino to com-
plex isoquinoline compounds. According to their structure,
precursor molecules and biosynthetic pathway, these com-
pounds can be classified in several types such as protoalk-
aloid, purine alkaloid, pyridine alkaloid, pseudo alkaloid,
quinoline, indole alkaloid, isoquinoline alkaloids, tropine
and psedotropine alkaloids (Guo et al., 2013). Alkaloids
have a great impact on human health related issues due
to their direct activity on nervous system for example caf-
feine in Tea and coffee. Some alkaloids have been reported
having psycotropic effect such as cannabin from Cannabis
sativa function as hallucinogen. Very few of them, only 1%,
were used as medicine, may be due to lack of significant
research for their extraction and isolation procedure as
these are synthesized in a very limited amount. So there
should be an alternative method to produce these mole-
cules in another system and this can be done with the aid
of comprehensive understanding of biochemical process
related to their synthesis. Very limited data are available
for the genome sequences of medicinal plants, so next gen-
eration sequencing is extensively used to construct the
metabolic pathway database of medicinally important non-
model plants (Wang et al., 2009). C. roseus is considered as
a model plant for alkaloid production, it is only source of
anticancer drugs vincristine and vinblastine (Jacobs et al.,
2005). Using RNA-seq data, reference transcriptome for
C. roseus, Cathcyc version 1.0, was generated, which is eas-
ily accessible online database (www.cathacyc.org) consist-
ing information about the metabolites and gene expression
data. This database is focused on specific secondary meta-
bolites (TIAs) of C. roseus. Almost all steps of the pathway
involved in synthesis of TIAs were illustrated by annotation
of sequences. Hypericum perforatum L. (St. John’s wort) is
another important medicinal plant having indole alkaloids
(serotonin and melatonin) along with hypericin and hyper-
forin as active ingredients. De novo transcriptome analysis
of H. perforatum showed that out of 40,813 annotated uni-
genes (68.86%), 2359 were assigned for secondary metab-
olism (He et al., 2012). Genes involved in camptothecin, a
terpenoid indole alkaloid, and biosynthesis was identified
through pyrosequencing of Camptotheca acuminata (Sun
et al., 2011).
Elucidation of phenylpropanoid pathway through
transcriptomics approach
Phenyl propanoids also constitute a large group of second-
ary metabolites and these help as a structural component,
in protection against biotic and abiotic stress through anti-
oxidative properties (Korkina et al., 2011). These are
synthesized via a very complex branching pathway, basic-
ally start with phenyl alanine and leads to coumaryl CoA.
First step of this pathway is catalyzed by phenyl alanine am-
monia lyase (PAL), designated as a rate limiting enzyme of
this pathway (Zhao et al., 2013). Several derivatives of phe-
nyl propanoid are reported such as flavanoids, isoflavo-
noids, coumarins and lignins. Ocimum species is
considered as a rich source of phenyl propanoids on the
basis of phytochemical analysis and recently de novo tran-
scriptome sequencing of Ocimum basilicum confirmed it
at genetic level (Rastogi et al., 2014). Genes involved in
phenyl propanoid pathway were elucidated using KEGG
analysis. It is expected that with the availability of transcrip-
tomic data sets for more of Ocimum species, more aspects
related with the diversity, origin and evolution of biosyn-
thetic pathway will be explored in near future.
Some other modifying enzymes involved in
secondary metabolism:
Besides the genes involved directly in biosynthesis path-
way of secondary metabolites , there is another group of
enzymes collectively known as modifying enzymes and ex-
amples of these include Cyp P450 gene family, GTs (gluco-
syl transferase), etc. (Tiwari et al., 2014; Srivastava et al.,
2015). The metabolome diversity is regulated by a diverse
group of enzymes, which mediate terminal transformations.
These are also extracted from transcriptome assembly and
categorized on the basis of their substrate specificity. In
case of A. indica attempts have located specific cytochrome
P450, which are putative candidates involved in neem iso-
prenoid pathway (Rajakani et al., 2014).
Molecular markers
An important contribution of next generation sequencing
is the identification of molecular markers in non-model
organisms (Marshall et al., 2003; Parchman et al., 2010).
Identification of molecular markers using transcriptome ap-
proach is very cost effective method in comparison with
conventional plant breeding methods. Molecular markers
such as Single sequence repeat (SSR) and SNP can be iden-
tified using transcriptome data and these can be further
Medicinal plant transcriptomes
used for genetic diversity analysis, quantitative trait loci
(QTL), marker assisted selection, gene flow, parental ana-
lysis and in evolutionary studies (Thiel et al., 2003; Egan
et al., 2012; Liu et al., 2013; Zheng et al., 2013). This was
followed in transcriptome analysis of many medicinal
plants for example in C. asiatica (Sangwan et al., 2013),
W. somnifera (Gupta et al., 2013). In W. somnifera di-
nucleotide SSRs are highest in number (2489) followed
by trinucleotide (1681), tetra (92) and then penta
(20)-nucleotide.
Identification of transcription factors (TFs):
TFs are cis regulatory elements and these are generally
DNA binding protein in nature. Basic feature of TF(s) is
the presence of DNA binding domain that provides access
to bind up with the trans regulatory region on DNA. TF(s)
can regulate the expression of gene i.e. which gene will be
switch on or off in a particular condition enabling complex
eukaryotic genome of plant to operate in a well-defined
and controlled manner for the expression of genes. TF(s)
play a diverse role in plant immunity as these helps in sig-
nalling in different environmental conditions, at different
developmental stages, during stress condition and in de-
fence responses through interaction towards plant patho-
gen (VomEndt et al., 2002). These TF(s) have a critical
role in manipulation of secondary metabolic pathways be-
cause single TF may be able to modulate the whole meta-
bolic pathway by switching on and off of genes. Thus,
identification and isolation of TF(s) controlling metabolic
pathways through transcriptome analysis is more dynamic
approach (Table 3). Most commonly identified classes of
TFs are WRKY, MYB, Zinc finger family, F-Box Homeobox,
WD40 repeat family, bHLH, pathogenesis-related/ERF and
AP2, etc (Xie et al., 2014). While analysing transcriptome
Table 3. List of transcription factors commonly involved in secondary metabolism
Transcription factor Plant name
WRKY1 Gossypium arboreum
R2R3-MYB Vitis vinifera
MYB11/R2R3-MYB Arabidopsis thaliana
MYB Antirrhinum majus
DOF4;2/C2C2-DOF Arabidopsis thaliana
MYB10 apple (Malus × domestica),
ODO1/R2R3-MYB Petunia hybrida
ORCA (AP2/ERF-domain) Catharanthus roseus
ATR1 (MYB34)/R2R3-MYB Arabidopsis thaliana
NST3/NAC Arabidopsis thaliana
PAP1/PAP2 (R2R3-MYB) Arabidopsis thaliana
P1 (R2R3-MYB) Zea maize
9
of C. asiatica, TFs belonging to 71 different families were
identified and among them major part was covered by Zinc
finger family (Sangwan et al., 2013). Besides, Zinc finger
other important classes were also reported such as F-box
Homeobox, bHLH, AP2, GRAS, GATA, MYB, WD40, etc.
TFs are involved in many developmental processes includ-
ing growth, development and adaptation therefore their se-
quence information can be applied to significantly utilize
their function for revealing their interaction with the pathway
genes. RNA sequencing i.e. transcriptome study was also
done in case of Solanum lycopersicum and a pool of TFs in-
volved in terpene synthesis was created, also a quantitative
expression database was obtained for jasmonic acid-treated
trichomes (Spyropoulou et al., 2014).
Comparative profiling of gene expression
Secondary metabolites are synthesized in particular plant
species as well as in specific tissue, so there may be a regu-
latory mechanism to control the biosynthesis of these me-
tabolites. Comparative transcript analysis through
transcriptomics data reveals that abundance of transcripts
is directly correlated with quantity of metabolites. For ex-
ample in W. somnifera leaf and root both differ in their me-
tabolites, these are further established with transcriptomic
data of W. somnifera leaf and root (Gupta et al., 2013,
2015; Sabir et al., 2013). Transcriptomic approaches can
be utilized in comparative profiling of gene expression ei-
ther in different tissue of same plant or between two differ-
ent species (Narnoliya et al., 2014; Garg et al., 2015).
Similarly in other plants such as A. annua, Mentha arven-
sis, etc. gene expression profiling has provided highly use-
ful information and could provide clues for future research
(Bose et al., 2013; Soetaert et al., 2013; Yadav et al., 2013,
2014). In case study of different species of Panax it was
Metabolic pathway/stress tolerance References
sesquiterpenoid aldehydes Xu et al. (2004)
phenylpropanoid pathway Deluc et al. (2006)
Flavonoids Stracke et al. (2001)
Growth and anthocyanins Waites et al. (1998)
Phenylpropanoids Skirycz et al. (2007)
Flavanoid biosynthesis Espley et al. (2007)
Benzenoids Verdonk et al. (2005)
Alkaloid pathway Van der Fits et al. (2001)
Indole-derived glucosinolates Celenza et al. (2005)
Lignin Mitsuda et al. (2005)
Anthocyanin biosynthesis Borevitz et al. (2000)
Phlobaphene/Flavonoids Grotewold et al. (1994)
10
revealed that 19,226 unique sequences of P. notoginseng
have similarity with P. quinquefolius but 11,626 transcripts
were specific to P. notogingseng. In comparison with P.
ginseng, in P. notoginseng 19,479 sequences were similar
and 11,373 unisequences were restricted to P. gingseng.
Identification of miRNA(s)
Furthermore miRNA may also be identified in transcrip-
tome data pool (Wu et al., 2012; Galla et al., 2013; Fan
et al., 2015; Prakash et al., 2016). In W. somnifera total
911 miRNA were identified by Das et al. (2014) in transcrip-
tome of salicylic acid treated leaf. Li et al. reported 14
miRNA from Panax gingseng. Recently high throughput
gene sequencing is being used to study the host–pathogen
interaction at genetic level. Dasgupta et al., 2014.
Conclusions and future prospects
The de novo assembly, annotation and analysis/investiga-
tion of transcriptome are one of the most prominent areas
of biological research going on worldwide. The simplicity
of tools and instrumentation along with cost affordability
has taken this technology to the doorstep of many labs
dealing with the biological researches. Thus, the area has
huge prospects in coming years in exploring and unravel-
ling the mysteries and intricacies of secondary metabolism.
Both the scientific aspects of investigations complement
each other very well and transcriptome assembly and ana-
lysis will tremendously assist in defining complex second-
ary metabolic pathways as well as in defining individual
steps. Currently, this is one of the most rapidly growing
areas and will continue to be so in near future until many
of the complex secondary metabolic pathways and their
regulation will be established and could be available to
be utilized in heterologous manner in synthetic biology
mode.
Acknowledgements
Authors are thankful to Network projects BSC203 and BSC
107 grants (Chembio and Plagen of CSIR), NMITLI (New
Millennium Indian Technology Leadership Initiative), and
DBT (Department of Biotechnology) projects for support-
ing various studies in the author’s laboratory, which has
generated knowledge and cited in the article. S. T. is thank-
ful to DST (Department of Science and Technology), New
Delhi for research support.
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