De novo transcriptome sequencing and metabolite profiling analyses reveal the complex metabolic genes involved in the terpenoid biosynthesis in Blue Anise Sage (Salvia guaranitica L.)

Abstract

Many terpenoid compounds have been extracted from different tissues of Salvia guaranitica. However, the molecular genetic basis of terpene biosynthesis pathways is virtually unknown. In this study, approximately 4 Gb of raw data were generated from the transcriptome of S. guaranitica leaves using Illumina HiSeq 2000 sequencing. After filtering and removing the adapter sequences from the raw data, the number of reads reached 32 million, comprising 186 million of high-quality nucleotide bases. A total of 61,400 unigenes were assembled de novo and annotated for establishing a valid database for studying terpenoid biosynthesis. We identified 267 unigenes that are putatively involved in terpenoid metabolism (including, 198 mevalonate and methyl-erythritol phosphate (MEP) pathways, terpenoid backbone biosynthesis genes and 69 terpene synthases genes). Moreover, three terpene synthase genes were studied for their functions in terpenoid biosynthesis by using transgenic Arabidopsis; most transgenic Arabidopsis 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 blue anise sage, and our study paves the way for the future metabolic engineering of the biosynthesis of useful terpene compounds in S. guaranitica.

Figure 1.

Typical GC-MS mass spectragraphs for terpenoids from old leaf, young leaf, stem, bud flower, flower and root of S. guaranitica. (A) GC-MS Peak of the essential oil, (B) mass spectrum of GC peak with retention time for the major compound, (C) Six-way Venn diagram to show the number of unique and common compounds in the essential oil extracts from old leaf (A), young leaf (B), stem (C), flower (D), bud flower (E) and root (F) of S. guaranitica.

Figure 2.

Functional annotation and classification of assembled unigenes in S. guaranitica. GO terms are summarized in three general sections of the BP, CC and 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 S. guaranitica transcriptome data with substrates and products, coloured arrows connect substrates to their corresponding products. Green/red colour-coded heat maps represent relative transcript levels of different terpenoid genes determined by Illumina HiSeq 2000 sequencing; red, up-regulated; green, down-regulated. Transcript levels data represent by FPKM: fragments per kilobase of transcripts per million mapped fragments. MeV, multi-experiment 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 diphosphate synthase; FPPS, farnesyl pyrophosphate synthase; GGPS, geranylgeranyl diphosphate synthase, type II; CINS, 1,8-cineole synthase; MYS, myrcene/ocimene synthase; LINS, (3S)-linalool synthase; NEOM, (+)-neomenthol dehydrogenase; SABI, (+)-sabinene synthase; TPS6, (-)-germacrene d synthase; AMS, beta-amyrin synthase; FARNESOL, farnesol dehydrogenase; SEQ, squalene monooxygenase; HUMS, α-humulene/β-caryophyllene synthase; FAR, farnesyl-diphosphate farnesyltransferase; 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-iso-kaurene 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. guaranitica. Total RNAs were extracted from old leaves, young leaves, stem, flower, bud flower and root samples and the expression of SgGPPS, SgFPPS, SgHUMS, SgNEOD-1, SgNEOD-2, SgNEOD-3, SgTPS-1, SgTPS-3, SgTPS-6, SgLINS-1, SgLINS-2, SgGLNS, SgGERIS, SgTPS-V and SgFARD genes were analysed using quantitative real-time. SgACTIN was used as the internal reference. The values are means ± SE of three biological replicates.

Figure 6.

Overexpression of three S. guaranitica terpenoid genes in transgenic Arabidopsis. (A) Comparison of the phenotypes of the transgenic A. thaliana and wild type A. thaliana. (B) Semiquantitative RT-PCR to confirm the expression of terpenoid genes.