Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds

Abstract Background Magnolia champaca, commonly known as champak is a well-known tree due to its highly fragrant flowers. Champak floral scent is attributed to a complex mix of volatile organic compounds (VOCs). These aromatic flowers are widely used in flavors and fragrances industry. Despite its commercial importance, the VOC biosynthesis pathways in these flowers are largely unknown. Here, we combine metabolite and RNA sequencing (RNA-seq) analyses of fully opened champak flowers to discover the active VOC biosynthesis pathways as well as floral scent-related genes. Results Volatile collection by headspace method and analysis by gas chromatography-mass spectrometry (GC-MS) identified a total of 43 VOCs from fully opened champak flowers, of which 46.9% were terpenoids, 38.9% were volatile esters and 5.2% belonged to phenylpropanoids/benzenoids. Sequencing and de novo assembly of champak flower transcriptome yielded 47,688 non-redundant unigenes. Transcriptome assembly was validated using standard polymerase chain reaction (PCR) based approach for randomly selected unigenes. The detailed profiles of VOCs led to the discovery of pathways and genes involved in floral scent biosynthesis from RNA-seq data. Analysis of expression levels of many floral-scent biosynthesis-related unigenes in flowers and leaves showed that most of them were expressed higher in flowers than in leaf tissues. Moreover, our metabolite-guided transcriptomics, in vitro and in vivo enzyme assays and transgenic studies identified (R)-linalool synthase that is essential for the production of major VOCs of champak flowers, (R)-linalool and linalool oxides. Conclusion As our study is the first report on transcriptome analysis of Magnolia champaca, this transcriptome dataset that serves as an important public information for functional genomics will not only facilitate better understanding of ecological functions of champak floral VOCs, but also provide biotechnological targets for sustainable production of champak floral scent

Additional file 1: Figure S1. of Integrated metabolome and transcriptome analysis of Magnolia champaca identifies biosynthetic pathways for floral volatile organic compounds

Quality and KEGG analysis of champak RNA-seq. Figure S2. Proposed biosynthesis pathways of volatile ester 2-methylbutanoate and its derivatives via the catabolism of branched-chain amino acid L-isoleucine. Figure S3. Comparison of deduced amino acid sequence of representative genes from pathways responsible for the production of VOCs. Figure S4. Phylogenetic analysis and amino acid alignment of champak DXSs. Figure S5. Amino acid sequence alignment of McHDR. Figure S6. Amino acid sequence alignment of McTPS1. Figure S7. GC-MS chiral analysis of β-linalool emitted from champak flowers. Figure S8. Analysis of transgenic N. tabacum overexpressing McTPS1. Table S1. MEP pathway genes from champak RNA-seq. Table S2. TPS genes from champak RNA-seq. Table S3. Accession numbers of proteins used in the TPS phylogenetic analysis. Table S4. List of primers used in this study. Table S5. Accession numbers of proteins used in the amino acid sequence alignments. Table S6. Accession numbers of proteins used in the DXS phylogenetic analysis. (PDF 1708 kb