RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula)

Abstract

Main conclusion

Using RNA-Seq, we cloned and characterized a unique monoterpene synthase responsible for the formation of a scent-determining S-linalool constituent of lavender oils from Lavandula × intermedia.

Several species of Lavandula produce essential oils (EOs) consisting mainly of monoterpenes including linalool, one of the most abundant and scent-determining oil constituents. Although R-linalool dominates the EOs of lavenders, varying amounts (depending on the species) of the S-linalool enantiomer can also be found in these plants. Despite its relatively low abundance, S-linalool contributes a sweet, pleasant scent and is an important constituent of lavender EOs. While several terpene synthase genes including R-linalool synthase have been cloned from lavenders many important terpene synthases including S-linalool synthase have not been described from these plants. In this study, we employed RNA-Seq and other complementary sequencing data to clone and functionally characterize the sparsely expressed S-linalool synthase cDNA (LiS-LINS) from Lavandula × intermedia. Recombinant LiS-LINS catalyzed the conversion of the universal monoterpene precursor geranyl diphosphate to S-linalool as the sole product. Intriguingly, LiS-LINS exhibited very low (~ 30%) sequence similarity to other Lavandula terpene synthases, including R-linalool synthase. However, the predicted 3D structure of this protein, including the composition and arrangement of amino acids at the active site, is highly homologous to known terpene synthase proteins. LiS-LINS transcripts were detected in flowers, but were much less abundant than those corresponding to LiR-LINS, paralleling enantiomeric composition of linalool in L. × intermedia oils. These data indicate that production of S-linalool is at least partially controlled at the level of transcription from LiS-LINS. The cloned LiS-LINS cDNA may be used to enhance oil composition in lavenders and other plants through metabolic engineering.

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Abbreviations

EOs:

Essential oils

GPP:

Geranyl pyrophosphate

mTPS:

Monoterpene synthase

LiS-LINS:

L. × intermedia S-linalool synthase

LiR-LINS:

L. × intermedia R-linalool synthase

NPP:

Neryl pyrophosphate,

qPCR:

Quantitative real-time PCR

TPS:

Terpene synthase

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Acknowledgements

This work was supported through Grants and/or in-kind contributions to SSM by UBC, and Natural Sciences and Engineering Research Council of Canada. We thank Dr I. Hwang, Pohang University of Science and Technology, Korea for kindly providing p326::sGFP construct DNA; Aaron Johnstone, UBC, Dr. Phillip Barker’s lab for assisting on inverted fluorescent microscopy and Joan Chisholm, Dr. Helene Sanfacon’s Lab, Summerland Agriculture and Agri-Food Canada for assisting on N. benthamiana protoplasts isolation and transfection.

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Correspondence to Soheil S. Mahmoud.

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Appendix S1: Annotated transcriptome sequences from Lavandula (XLSX 5066 kb)

Appendix S2: KEGG pathway distribution using KO annotation (XLSX 19 kb)

Table S1. TPSs clustered under TPS-g subfamily, and previously known from Lavandula, with the corresponding GenBank/ UniProt accession numbers that are incorporated in the phylogenetic analysis of LiS-LINS (DOCX 13 kb)

Table S2. Species distribution of the top BLAST hits of Lavandula transcriptome unigenes. The transcriptomic sequences from Lavandula were highly homologous to Solanum lycopersicum (23.59 %) and Vitis vinifera (15.45 %) compared to transcriptome sequences from any of other plant species (< 8 %) (DOCX 12 kb)

Table S3. Summary of the most common protein families and domains found in the Lavandula transcriptome database. Of the total protein classes and domains detected from Lavandula transcriptome sequences, protein kinases, nucleoside triphosphate hydrolase and Leucine-rich repeat domain are the most abundant containing over 1000 copy numbers (DOCX 16 kb)

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Adal, A.M., Sarker, L.S., Malli, R.P.N. et al. RNA-Seq in the discovery of a sparsely expressed scent-determining monoterpene synthase in lavender (Lavandula). Planta 249, 271–290 (2019). https://doi.org/10.1007/s00425-018-2935-5

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Keywords

  • Essential oils
  • Lavandula
  • S-Linalool
  • S-Linalool synthase
  • RNA-Seq
  • Terpene synthase