Transcriptome and metabolite analyses reveal the complex metabolic genes involved in volatile terpenoid biosynthesis in garden sage (Salvia officinalis)

Abstract

A large number of terpenoid compounds have been extracted from different tissues of S. officinalis. However, the molecular genetic basis of terpene biosynthesis pathways is virtually unknown. In this study, approximately 6.6 Gb of raw data were generated from the transcriptome of S. officinalis leaves using Illumina HiSeq 2000 sequencing. After filtering and removing the adapter sequences from the raw data, the number of reads reached 21 million, comprising 98 million of high-quality nucleotide bases. 48,671 unigenes were assembled de novo and annotated for establishing a valid database for studying terpenoid biosynthesis. We identified 135 unigenes that are putatively involved in terpenoid metabolism, including 70 mevalonate and methyl-erythritol phosphate pathways, terpenoid backbone biosynthesis genes, and 65 terpene synthase genes. Moreover, five terpene synthase genes were studied for their functions in terpenoid biosynthesis by using transgenic tobacco; most transgenic tobacco plants expressing these terpene synthetic genes produced increased amounts of terpenoids compared with wild-type control. The combined data analyses from the transcriptome and metabolome provide new insights into our understanding of the complex metabolic genes in terpenoid-rich sage, and our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. officinalis.

Figure 1

Typical GC-MS mass spectragraphs for terpenoids from young leaf, old leaf, and stem of Salvia officinalis. (A) GC-MS peaks of the essential oil extracts, (B) Mass spectrum of GC peaks with retention time for the major compound. (C) Three-Way-Venn-Diagram to show the number of unique and common compounds in the essential oil extracts from young leaf (A), old leaf (B), and stem (C) of Salvia officinalis.

Figure 2

Functional annotation and classification of assembled unigenes from S. officinalis. Gene Ontology (GO) terms are summarized in three general sections of the biological process (BP), cellular component (CC) and molecular function (MF).

Figure 3

KEGG classified into five largest categories pathways includes cellular processes (A), environmental information processing (B), genetic information processing (C), metabolism (D) and organismal systems (E).

Figure 4

Representative terpenoid biosynthesis pathway with cognate heat maps for transcript levels of genes from transcriptome data with substrates and products, colored arrows connect substrates to their corresponding products. Green/red color-coded heat maps represent relative transcript levels of different terpenoid genes determined by Illumina HiSeq 2000 sequencing; red, upregulated; green, downregulated. Transcript levels data represent by FPKM: Fragments per Kilobase of transcripts per Million mapped fragments. MeV: MultiExperiment Viewer software was used to depict transcript levels. DXS: 1-deoxy-D-xylulose-5-phosphate synthase, DXR:1-deoxy-D-xylulose-5-phosphate reductoisomerase, MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, ISPF: 2-C-methyl-D-erythritol 2,4-cyclodiphos-phate synthase, HDS:(E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase, HDR: 4-hydroxy-3-methylbut-2-enyl diphosphate reductases, IDI: isopentenyl-diphosphate delta isomerase, AACT: acetyl-CoA C-acetyl transferase, HMGS: hydroxyl methyl glutaryl-CoA synthase, HMGR: hydroxymethyl glutaryl-CoA reductase (NADPH), MVK: mevalonate kinase, PMK: phospho-mevalonate kinase, GPPS: geranyl pyrophosphate synthase, FPPS: farnesyl pyrophosphate synthase, GGPS: geranylgeranyl pyrophosphate synthase, type II, CINO:1,8-cineole synthase, MYS: myrcene/ocimene synthase, LINA: (3S)-linalool synthase, NEOM:(+)-neomenthol dehydrogenase, SABI:(+)-sabinene synthase, TPS6:(−)-germacrene D synthase, AMS:beta-amyrin synthase, SEQ: Squalene monooxygenase, HUMS:α-humulene/β-caryophyllene synthase, GA2:gibberellin 2- -oxidase, GA20:gibberellin 20-oxidase, E-KS:ent-kaurene synthase, MAS:momilactone-A synthase, GA3:gibberellin 3-beta-dioxygenase, E-KIA: ent-isokaurene C2-hydroxylase, E-KIH:ent-kaurenoic acid hydroxylase, E-CDS: ent-copalyl diphosphate synthase.

Figure 5

Quantitative RT-PCR validation of expression of terpene synthase genes selected from the DGE analysis in S. officinalis. Total RNAs were extracted from young leaves, old leaves, stem, flower and bud flower samples and the expression of SoNEOD, SoGPS, SoFPPS, SoGGPS, SoMYRS, SoLINS, SoHUMS, SoTPS6, SoSQUS, SoSABS and SoCINS genes were analysed using quantitative real-time. SoACTIN was used as the internal reference. The values are means ± SE of three biological replicates.

Figure 6

Overexpression of five S. officinalis TPS genes in transgenic N. tabacum. (A) Transgenic tobacco plants after adaptation to soil pots. (B) Semiquantitative RT-PCR analysis of the terpene synthase gene expression.

Figure 7

Phylogenetic analysis of terpenoid biosynthesis genes from S. officinalis and other plants. MEGA6 program was used for building up the tree through neighbor joining method.