Engineering storage capacity for volatile sesquiterpenes in Nicotiana benthamiana leaves.

Thierry L Delatte,Leticia Alves Gomes Albertti,Carlos Carollo,Jules Beekwilder,Jacqueline Busscher,Francel Verstappen,Jamil Molenaar,Katyuce De Sousa Farias,Harro Bouwmeester,Giulia Scaiola

doi:10.1111/pbi.12933

Thierry L Delatte, Leticia Alves Gomes Albertti + Show 8 more

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https://doi.org/10.1111/pbi.12933

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Abstract

SummaryPlants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often carried out in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In this study, we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin‐coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil seed‐producing species castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co‐expression of α‐bisabolol synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from castor bean promoted storage of α‐bisabolol in N. benthamiana mesophyll tissue more than 17‐fold. A clear correlation was found between neutral lipids and storage of sesquiterpenes, using synthases for α‐bisabolol, (E)‐β‐caryophyllene and α‐barbatene. The co‐localization of neutral lipids and α‐bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants.

Highlights

Sesquiterpenes form a structurally diverse family of hydrophobic molecules
This research was initiated when a construct expressing the Arabidopsis (E)-b-caryophyllene synthase AtTPS21 using the 35S promoter was introduced in Arabidopsis thaliana (Columbia 0), and seeds from confirmed transformed plants were analysed by GC-MS (Ting et al, 2015)
The lipid content of Arabidopsis seeds of the Columbia 0 ecotype is 35% (w/w) (Li et al, 2006). This suggests that lipid bodies naturally present in plant tissues can function as storage for sesquiterpenes

Summary

Introduction

Sesquiterpenes form a structurally diverse family of hydrophobic molecules. They consist of three isoprene units concatenated by the farnesyl diphosphate synthase in to the sesquiterpene precursor farnesyl diphosphate (FPP). Plant-produced sesquiterpenes are used as ingredients in pharmaceutical, cosmetic and flavouring products. The commercial exploitation of natural terpene-containing plant sources requires sustainable plant cultivation. This is challenging when the source plant accumulates the compound of interest in economically viable quantities only after a growth period of 10–15 years. Unregulated collection of such plants may endanger their wild populations and ecosystems. As a consequence of overexploitation of the naturally occurring populations, Candeia material is no longer sufficiently available to meet market demands for a-bisabolol, and alternative sources for this compound are under investigation (Han et al, 2016; de Meireles et al, 2015; Son et al, 2014)

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