De novo transcriptome analysis of rose-scented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis

Narnoliya et al. BMC Genomics (2017) 18:74 DOI 10.1186/s12864-016-3437-0 RESEARCH ARTICLE Open Access De novo transcriptome analysis of rosescented geranium provides insights into the metabolic specificity of terpene and tartaric acid biosynthesis Lokesh K. Narnoliya, Girija Kaushal, Sudhir P. Singh* and Rajender S. Sangwan* Abstract Background: Rose-scented geranium (Pelargonium sp.) is a perennial herb that produces a high value essential oil of fragrant significance due to the characteristic compositional blend of rose-oxide and acyclic monoterpenoids in foliage. Recently, the plant has also been shown to produce tartaric acid in leaf tissues. Rose-scented geranium represents top-tier cash crop in terms of economic returns and significance of the plant and plant products. However, there has hardly been any study on its metabolism and functional genomics, nor any genomic expression dataset resource is available in public domain. Therefore, to begin the gains in molecular understanding of specialized metabolic pathways of the plant, de novo sequencing of rose-scented geranium leaf transcriptome, transcript assembly, annotation, expression profiling as well as their validation were carried out. Results: De novo transcriptome analysis resulted a total of 78,943 unique contigs (average length: 623 bp, and N50 length: 752 bp) from 15.44 million high quality raw reads. In silico functional annotation led to the identification of several putative genes representing terpene, ascorbic acid and tartaric acid biosynthetic pathways, hormone metabolism, and transcription factors. Additionally, a total of 6,040 simple sequence repeat (SSR) motifs were identified in 6.8% of the expressed transcripts. The highest frequency of SSR was of tri-nucleotides (50%). Further, transcriptome assembly was validated for randomly selected putative genes by standard PCR-based approach. In silico expression profile of assembled contigs were validated by real-time PCR analysis of selected transcripts. Conclusion: Being the first report on transcriptome analysis of rose-scented geranium the data sets and the leads and directions reflected in this investigation will serve as a foundation for pursuing and understanding molecular aspects of its biology, and specialized metabolic pathways, metabolic engineering, genetic diversity as well as molecular breeding. Keywords: Rose-scented geranium, Pelargonium sp. cv. Bourbon, De novo transcriptome, Terpene, Tartaric acid, Ascorbic acid, Anacardic acid Background Rose-scented geranium (Pelargonium sp.) is a perennial aromatic and medicinal herb of family Geraniaceae. The genus Pelargonium contains about 750 species growing in temperate and subtropical climate [1]. Most of them were indigenous to South Africa, introduced in Europe * Correspondence: ; ; Center of Innovative and Applied Bioprocessing (A National Institute under the Department of Biotechnology, Govt. of India), S.A.S. Nagar, Mohali, Punjab, India during 17th century, and subsequently spread all over the world [2, 3]. Aroma possessing species of geranium, such as P. graveolens (synonym-P. roseum), has a history of folkloric significance. Aerial parts of rose-scented geranium have traditionally been used as insect repellent, perfume and flavouring agents, antimicrobial and aroma-therapeutic herb as well as medicinal plant material of advantage in gastrointestinal disorders, hyperglycemia, and healing [4, 5]. © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Narnoliya et al. BMC Genomics (2017) 18:74 The vegetative and reproductive aerial parts of rosescented geranium develop numerous epidermal emergences of glandular and non-glandular nature, known as trichomes [6]. The non-glandular trichomes, often unicellular, sometimes bicellular and rarely multicellular, could be physiologically beneficial to plants during temperature regulation, reduction of water loss and, metal tolerance. [7]. Glandular trichomes, the most numerous in leaves, are specialized tissues comprised of a basal stalk and a head of secretory cells that accumulate essential oils [6]. Essential oils are complex volatile compounds, such as terpenes, esters, alcohols, aldehydes, ketones, and phenols, produced in plants as bioactive secondary metabolites, often for ecological adjustment and protection from microbial pathogens, fungi, pests and predation [8]. The main constituents of essential oil of rose-scented geranium are acyclic monoterpenoids and acetate esters of monoterpenols [5]. The most abundant monoterpenoids are citronellol, geraniol, roseoxide, linalool, and citronellyl formate [9]. The antioxidant, antibacterial, antifungal, antiviral, antiseptic, antidiabetic, antihemorrhoids and antitumor activities of the essential oils and their constituents have been widely studied [1, 10]. The distillate and absolute extracts (essential oil) from the foliage of the herb have a pleasant rose-like fragrance, and therefore are used as a substitute of expensive rose oil [11]. Further, Geraniaceae plants have been reported to synthesize and accumulate tartaric acid in leaves, possibly by ascorbate metabolism [12, 13]. Natural tartaric acid is a food additive serving as antioxidant, leavening agent, and flavor enhancer. Our group has developed a process for the production of scented natural tartaric acid from rose-scented geranium biomass as well as from residual water after hydro-distillation of the herb [13]. Thus, rose-scented geranium is a cash crop of high significance in pharmaceutical, food, phytoremediation, sanitary, cosmetic and perfume industries [14, 15]. There have been fewer molecular and biochemical studies on rose-scented geranium due to limited gene sequence information, as only 9 and 4 sequences were encountered on search of public domain nucleotide and protein databases, respectively, in NCBI GenBank dated December 21, 2016 (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=73200). Moreover, biochemical studies on the plant have been lacking as the plant was recognized as a hyper-acidic one [15]. Sangwan et al. [16] provided a process for isolation of proteins and catalytically active enzymes from rose-scented geranium. Next-generation sequencing (NGS) technologies have accelerated transcriptome investigations in several plant species, exploring qualitative and quantitative insights of global gene regulation [17]. In SRA database, raw sequencing reads a (...truncated)