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

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: Anacardic acid; Ascorbic acid; De novo transcriptome; Pelargonium sp. cv. Bourbon; Rose-scented geranium; Tartaric acid; Terpene.

Fig. 1

Field grown rose-scented geranium

Fig. 2

Distribution of rose-scented geranium contigs according to their size

Fig. 3

Distribution of the top hits for unique proteins in NR database

Fig. 4

Schematic representation of terpene biosynthetic pathway, and heatmaps displaying the expression (log2 FPKM) of enzymes involved in the different reaction steps. The details of the transcripts are given in Additional file 6. AACT, acetoacetyl-CoA thiolase/acetyl-CoA acetyltransferase; HMGS, hydroxymethylglutaryl- CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; 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; CMK, 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase; MDS, 2-C-methyl- D -erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphatereductase; GPPS, geranyl diphosphate synthase; IDI, isopentenyl-diphosphate delta isomerase; FPPS, farnesyl pyrophosphate synthase; GGPPS, geranylgeranyl diphosphate synthase; MTPS, mono-terpene synthase; STPS, sesqui-terpene synthase; DTPS, di-terpene synthase; HMG, CoA, hydroxymethylglutaryl-CoA; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GA-3P, glyceraldehyde 3-phosphate; DXOP, 1-deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-d-erythritol-phosphate; CDP-ME, 4-(cytidine5′ -diphospho)-2-C-methyl-d-erythritol; CDP-ME2P, 2-phospho 4- (cytidine 5′-diphospho)2-c-methyl-d-erythritol; ME 2,4 cPP, C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; MVA, mevalonic acid

Fig. 5

Schematic representations of ascorbic acid and tartaric acid biosynthesis, and heatmaps displaying the expressed transcripts (log2 FPKM) related to enzymes involved in the different reaction steps. Transcripts were not detected for the enzymes represented in gray color. The details of the transcripts are given in Additional file 7

Fig. 6

Schematic representation of anacardic acid biosynthesis, and heatmaps displaying the expressed transcripts (log2 FPKM) related to enzymes involved in the different reaction steps. The details of the transcripts are given in Additional file 8

Fig. 7

Putative orthologous TF genes (>10) belonging to different TF families (a), and putative TF genes regulating terpene biosynthesis (b). The details of the transcripts are given in Additional file 9

Fig. 8

Putative orthologous genes related to different hormones. The details of the transcripts are given in Additional file 10

Fig. 9

Quantitative real time PCR analysis (a) to validate FPKM expression values (b) of randomly selected contigs from rose-scented geranium leaf transcriptome. DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; GPPS, geranyldiphosphate synthase; FPPS, farnesyl pyrophosphate synthase; LS, linalool synthase; HK, hexokinase; ME, GDP-mannose-3′, 5′ -epimerase; IDH, L-idonate 5-dehydrogenase; PG, polygalacturonase; WRKY, WRKY DNA binding protein 4; MYB; ZE, zeaxanthin epoxidase; CYP, cytochrome P₄₅₀