Transcriptome analysis of distinct Lindera glauca tissues revealed the differences in the unigenes related to terpenoid biosynthesis

Highlights

• The roots, sarcocarps, stems, leaves and kernels of L. glauca were as materials.

• The first report of the Illumina transcriptome sequencing in L. glauca

• The unigene annotation and the analysis differential expression were performed.

• The unigenes involved in terpenoid biosynthesis were concretely discussed.

• The differences of terpenoid biosynthesis existed in distinct L. glauca tissues.

Abstract

The Lindera glauca, an economically and ecologically important tree species, has emerged as a novel potential plant for the intensive studies of essential oil owing to its characteristic aroma and medicinal property in distinct tissues. However, the transcriptome information and molecular research on this species is still unknown to date. To reveal the formation and accumulation mechanism of essential oil in distinct L. glauca tissues, it is crucial to analyze transcriptome and to identify the full repertoire of potential unigenes involved in terpenoid biosynthesis. In this paper, the transcriptomes of the roots, sarcocarps, stems, leaves and kernels of L. glauca were analyzed for the first time by using short-read sequencing technology (Illumina). A total of 27.2 GB valid reads (the average length = 92.7 bp) was obtained from distinct L. glauca tissues, and then assembled de novo into 264,831 unigenes by Trinity strategy (mean size = 560.2 bp). The resulting 98,141 unigenes (38%) of all the assembled unigenes were annotated in multiple public databases, of which 114 potential unigenes were identified to be involved in the terpenoid biosynthetic accumulation in L. glauca. Additionally, the differential expression profiles revealed 675, 697, 432, 1702 and 844 high tissue-specificity expressions of unigenes in the roots, sarcocarps, stems, leaves and kernels of L. glauca, respectively. Overall, these obtained comprehensive unigene resources will contribute to advance the research regarding the specific plant and more specifically discovery of genes participating in the terpenoid pathway and its regulation in specific tissues of the L. glauca, but also could help the understanding of the differential accumulation of secondary metabolites in distinct plant tissues.

Introduction

The terpenoids (or isoprenoids), derived from isopentenyl diphosphate (IPP, C5) and its isomer dimethylallyl diphosphate (DMAPP, C5), constitute one of the most structurally diverse groups of natural compounds, and over 55,000 different terpenoids have been isolated (Breitmaier, 2006). In higher plants, there are two known biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway (Li et al., 2008), in which the IPP and DMAPP are produced as the precursors for sesquiterpenes, triterpenes, sterols and polyterpenes in cytoplasm, and for monoterpenes, diterpenes and tetraterpenes in plastid pool (Gershenzon and Dudareva, 2007). It is generally accepted that the terpenoids exist widely in living organisms owing to its biological function diversity such as photosynthesis, respiration, growth, cell cycle control, plant defense and environmental adaptation (Estévez et al., 2001). Also, the industrial and medical values of the plant terpenoids have been reported as the important raw materials for flavors, fragrances and spices as well as medicines against cancer, malaria, inflammation and a variety of infectious diseases (viral and bacterial) (Zhao et al., 2014, Sangwan and Sangwan, 2014, Wang et al., 2005). Thus, a detailed understanding of terpenoid biosynthetic mechanism is the prerequisite for fully terpenoid utilization.

Lindera glauca (Lauraceae), a pretty and small deciduous tree, is extensively distributed in the mountainous districts of China, Japan and Korea (Wang et al., 1994). In China, this plant has emerged as one of the most economically and ecologically important and intensively studied tree species owing to its very plentiful resource, well developed roots, superior adaptability and wide application in essence and perfume, medicine and chemical industry (Wang et al., 1994, Liu et al., 1992, Sun et al., 2011). Generally, the fruits, roots and leaves of L. glauca have been traditionally used as a type folk medicine for the extensive treatment of several symptoms of disease in Asia. For instance, N-methyllaurotetanine, N-trans-feruloyltyramine and N-cis-feruloyltyramine from L. glauca exhibited significantly anti-tumor metastatic activities (Wang et al., 2011), and some antioxidant flavonoids have been identified from L. glauca heartwood (Huh et al., 2014). Moreover, the volatile oil from the leaves and fruits of this plant has been reported to show very strong antimicrobial activity to the tested bacteria, fungi, mold or yeast in vitro (Liu et al., 1992, Sun et al., 2011). Besides those medicinal properties, the isolated volatile constituents of L. glauca leaves mainly which contain β-phellandrene (19.03%), myrcene (17.93%), aromadendrene (17.11%), γ-cadinene (10.17%) and allo-ocimene (9.17%) have been reported (Liu et al., 1992), whereas ocimene (31.90%), copaene (12.75%), and α-caryophyllene (8.06%) were identified as the main compounds in L. glauca fruits (Sun et al., 2011). It is instructive to note that there is an obvious variance of the volatile constituents and its main compounds among different L. glauca tissues. Although significant progress has been made in chemical components and pharmacological effects of essential oils from the leaves, roots and fruits of L. glauca (Wang et al., 1994, Liu et al., 1992, Sun et al., 2011, Wang et al., 2011, Huh et al., 2014, Seki et al., 1994, Huh et al., 2012), up to now the reports about the biochemical and molecular mechanisms of the terpenoid biosynthesis in this plant remain very limited, especially in transcriptome and gene expression analysis. Moreover, the lack of transcriptomic information and genomic resources (only 68 nucleotide sequences had been deposited in the NCBI GenBank database prior to 37 March 2014) is the bottleneck for further studies of biosynthetic mechanisms of the terpenoids in non-model plant L. glauca, thus the transcriptomic analysis of L. glauca is imperative.

Recently, advances in molecular biology and decreasing cost of next-generation sequencing technology make RNA sequencing (RNA-seq) to become a more effective choice for the transcriptomic studies especially in non-model species (Dillies et al., 2013). Consequently, RNA-seq has been widely deployed in many species, such as goose (Xu et al., 2013), Salvia miltiorrhiza (Yang et al., 2013) and Liriodendron chinense (Yang et al., 2014), and in many fields of research, including detection of alternative splicing isoforms (Wang et al., 2008), de novo assembly of transcripts (Xu et al., 2013), strand-specific expression (Levin et al., 2010). However, to date the study of L. glauca transcriptome is not reported in the world.

Here, we report on the Illumina transcriptome sequencing, functional annotation and differential expression profiles in the five distinct tissues (root, sarcocarp, stem, leaf and kernel) of L. glauca, which will provide a useful resource for gene excavation, molecular marker development and genetic improvement in L. glauca. Additionally, to further explore the differences of candidate unigenes in terpenoid biosynthesis among these distinct L. glauca tissues, the transcriptional levels of all the related-unigenes were concretely discussed. The results from our works could contribute to the discovery of genes complicated in the terpenoid pathway and its accumulative regulation of volatile constituents in specific tissues of L. glauca. To our knowledge, this is the first report of de novo transcriptome analysis in L. glauca to reveal the genes involved in terpenoid biosynthesis.