Podophyllum spp. - an endangered, perennial medicinal herb, is native to eastern North America and Himalayas. Medicinal plants are a vital source of several specialized, organic compounds called secondary metabolites. These compounds are widely used in pharmaceutical industries for their therapeutic potential. The biosynthetic pathways of these secondary metabolites are quite complex. Next-generation sequencing (NGS) has accelerated research opportunities in medicinal plants whose genome sequences are not available. In recent past, NGS technologies have generated a large number of transcriptome data in several economically important medicinal plants. NGS has also paved the way of exploring genomes of various endangered medicinal plants and this will in turn help us to develop conservation strategies to protect these valuable plants. In the present review, we have briefly discussed NGS research on various medicinal plants with special emphasis on the anticancer herb - Podophyllum spp. NGS methods play an important role in discovering genes that are involved in secondary metabolites biosynthesis. Identification of pathway genes is necessary for metabolic engineering of medicinal plants for the production of improved secondary metabolites.

• Podophyllotoxin; Transcriptome sequencing; Endangered; Secondary metabolites

Biswas S, Hazra S, Chattopadhyay S (2018) A Brief Outline of Next-Generation Sequencing Research on Podophyllum spp. – an Endangered Medicinal Herb. JSM Environ Sci Ecol 6(2): 1064.

PTOX: Podophyllotoxin; NGS: Next-Generation Sequencing; RNA-Seq: RNA Sequencing; KEGG: Kyoto Encyclopedia of Genes and Genomes; IUCN: The International Union for Conservation of Nature; miRNA: micro Ribonucleic Acid

Plant genomics - a recently evolved field of plant sciences, provides valuable information of structures, organizations and functions of plant genome. It also helps us to understand the molecular network of genes which are involved in the growth and development of plants. Next-generation sequencing (NGS), also known as massively parallel sequencing, can generate millions to billions of reads in a single instrument run. In comparison to Sanger sequencing [1], which is considered a first-generation DNA sequencing technology, NGS technologies has efficaciously reduced the cost and sequencing time. NGS methods have some significant advantages over the firstgeneration sequencing technology. First, NGS do not require arduous bacterial cloning method; here the sequencing libraries are clonally amplified in vitro. Second, Sanger sequencing utilizes chain termination chemistry whereas, NGS method is based on the addition of nucleotides to the complementary strand. Lastly, NGS methods perform sequencing in an immense parallel fashion. The commonly used platforms of NGS technologies are Roche 454 pyro-sequencing, Illumina, Ion Torrent, PacBio and SOLiD. Next generation RNA sequencing (RNA-seq) is a highly advanced approach to study the cellular transcriptome. RNA-seq is rapidly replacing the microarray technique because, unlike the microarray method, RNA-seq can assemble the reads de novo without the need of mapping to reference genome.

A brief account of high-throughput sequencing in medicinal plants

NGS has opened a new era in plant genomics by genetically improving the crop plants and identifying biosynthetic pathways of various secondary metabolites. Plants produce a variety of specialized compounds called secondary metabolites (Alkaloids, Terpenoids, and Phenolic compounds). Plant secondary metabolites are widely used as pharmaceuticals, flavourings, fragrances and industrial materials. These compounds are mostly found in medicinal plants for which genomic sequences are yet not available [2]. The de novo transcriptome sequencing has helped to identify the important pathway genes that are involved in the biosynthesis of secondary metabolites. P. ginseng is an important medicinal herb that produces ginsenoside. Research shows that ginsenoside have anticancer, antioxidative, vasorelaxation and anti-inflammatory properties. 454 pyrosequencing of P. ginseng roots discovered putative genes that are involved in ginsenoside biosynthesis [3,4]. Another valuable plant is Withania somnifera. It synthesizes withanolides which have high medicinal value. Transcriptome sequencing using 454-GS FLX platform detected genes which are involved in withanolide biosynthetic pathway [5]. NGS has also been successfully used to unravel the roles of pathway genes in Glycyrrhiza uralensis, Ginkgo biloba and Papaver somniferum [6,7,8]. Illumina Miseq platform was used to sequence Phyllanthus amarus leaf transcriptome and the study revealed the role of various pathway genes in secondary metabolites biosynthesis [9]. Recently, deep sequencing has aided the identification of genes involved in secoiridoid biosynthesis in Swertia mussotii, a Tibetan medicinal plant [10].

Glimpses of NGS research in Podophyllum spp. - an endangered medicinal herb

Podophyllum species are sources of an aryl tetralin lignan called podophyllotoxin. Podophyllotoxin is effective against many diseases, and is widely used as a starting compound for the semi-synthesis of anticancer drugs i.e. etoposide (VP-16-213), teniposide (VM-26) and etopophos [11,12]. The ever-growing demand has lead to habitat destruction and overexploitation of this medicinal plant and it is currently in the IUCN endangered list. Next generation high throughput sequencing has paved the way of identifying the genes that are involved in the podophyllotoxin biosynthesis. Recently, many works has been done in order to characterize PTOX metabolic pathway. Illumina transcriptome sequencing identified two cytochrome P450 enzymes, CYP719A23 from Podophyllum hexandrum and CYP719A24 from Podophyllum peltatum. Both of these enzymes can covert matairesinol into pluviatolide, ultimately leading to podophyllotoxin biosynthesis [13]. Additional studies using multi-omics approach (transcriptomics, metabolomics and bioinformatics) aided the discovery of aporphine alkaloid pathway in Podophyllum species and suggested evolutionary linkages between both lignan and alkaloid biosynthetic pathways [14]. 454 pyrosequencing of P. hexandrum cell culture transcriptome unravelled the roles of pathways genes in PTOX biosynthesis [15]. De novo transcriptome sequencing of Sinopodophyllum hexandrum at two temperatures (15°C and 25°C) was done using Illumina platform. This study showed up-regulation of phenylpropanoid pathway genes, ABA biosynthesis, ethylene & jasmonic acid at 15°C and the genes involved in stress tolerance were expressed at 25°C [16]. Lately, six novel enzymes have been identified in P. hexandrum. Among these enzymes, O-methyltransferases3 (OMT3) catalyzes methylation of pluviatolide to generate (–)-5′-desmethoxyyatein. This (–)-5′-desmethoxy-yatein is then further converted to (–)-4′-desmethylepipodophyllotoxin (the etoposide aglycone), that is the immediate precursor of etoposide [17], but the final steps for podophyllotoxin biosynthesis are yet to be characterized. Our group used 454 GS-FLX titanium pyrosequencing to identify the major transcripts of PTOX biosynthesis from MeJA treated P. hexandrum cell suspension culture. This study showed that PTOX biosynthetic pathway gene like PAL was minutely affected by MeJA treatment, whereas CCR, HCT, 4CL, CAD, cytochrome P450 and SAM-dependent methyltransferase were notably upregulated in treated condition [18]. A recent transcriptomic study provides deeper understanding of the mechanism that enhances plant growth and PTOX accumulation in S. hexandrum under higher elevation conditions. This study identified differentially expressed genes (DEGs) involved in PTOX biosynthesis, including phenylpropanoid enzymes: phenylalanine ammonia lyase (PAL), cinnamyl alcohol-dehydrogenase (CAD), dirigent protein (DIR), pinoresinol-lariciresinol reductase (PLR), CYP719A23, CYP71CU1, and 2-oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD) which directly participate in PTOX biosynthetic pathway [19].

In plants, miRNAs play pivotal roles in leaf development [20,21], anther development [22] and regulation of flowering time [23]. It also regulates secondary metabolites biosynthesis [24,25-27]. Several miRNAs has been identified in different plant species, but there are only few reports available on miRNA research in P. hexandrum. Our group identified conserved miRNAs and their putative target genes in P. hexandrum. KEGG analysis of these target genes showed that they are involved in various secondary metabolite biosynthetic pathways i.e. flavonoid, isoflavonoid, terpenoid, phenylpropanoid etc [28]. In another work from our group, we found five ROS non-responsive miRNAs that were significantly down regulated after MeJA treatment in P. hexandrum. These five miRNAs hindered expression of target genes, namely, 4-coumarate ligage (4CL), Cytochrome P450, Caffeoyl–CoA methyltransferase, and Chalclone synthase and the decline in the level of these miRNAs may bring about an up-regulation of the target genes [29]. In a recent work, six conserved miRNAs were identified in NGS transcriptomes of P. hexandrum. Detailed analysis showed that the target mRNAs viz. UDP glycosyltransferase, flavonol synthase, glyceraldehyde 3-phosphate dehydrogenase, peroxidase, malate dehydrogenase, phosphoenolpyruvate carboxylase, WRKY 37 and MYBF1 transcription factors are associated with secondary metabolites biosynthesis [30].

In the past decade, next-generation sequencing has changed the scenario of genomic research in plants. It has significantly saved the cost and time of sequencing. Furthermore, NGS is a powerful tool to discover genes/gene families that are associated with secondary metabolite biosynthetic pathways. The transcriptomic data available for medicinal plants are much less than the data available for crop and model plants. NGS methods have helped the scientists to generate biologically important data for valuable medicinal plants. De novo sequencing has played a vital role in discovering pathway genes without foregoing sequence knowledge.

The anticancer property of podophyllotoxin has raised its demand worldwide, but there is a scarce in raw materials and the in vitro production of PTOX is extremely low to meet its industrial demands. Therefore, to increase the overall yield of podophyllotoxin, alternative ways i.e. metabolic engineering needs to be explored. Since the biosynthetic pathway of PTOX is still not fully known, genetic intervention strategy is a strenuous process. Massively parallel sequencing has been successfully applied to discover the pathway genes in PTOX biosynthesis.

Till date, many works have been done to characterize the PTOX biosynthetic pathway. Our group identified potential genes and transcription factors involved in PTOX biosynthesis, thus providing valuable information for future investigation. Furthermore, we characterized PTOX specific CAD isoforms along with several candidate phenylpropanoid pathway genes which provides an insight into the molecular mechanism of PTOX biosynthesis. A recent report has also identified six novel enzymes that completes the pathway up to (–)-4′-desmethylepipodophyllotoxin, but not podophyllotoxin. Together, it can be envisaged that further in depth studies using NGS technologies would be highly beneficial to reconstitute the biosynthetic pathway up to PTOX [31].

The authors declare that there is no conflict of interest.

The authors are highly grateful to the Director, CSIR-IICB, for providing infrastructural facilities. This work was financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi. SB acknowledges the Department of Biotechnology (DBT), New Delhi for the fellowship.

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