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.

Plants produce a wide range of secondary metabolites. Within a single species, chemotypes can be distinguished by the differences in the composition of the secondary metabolites. Herein, we evaluated Nectandra megapotamica (Spreng.) chemotypes and the balance of different classes of metabolites to verify how significant differences in plant metabolism are regarding chemotypes. We collected N. megapotamica leaves from eight adult plants in two Brazilian states. The essential oils and ethanol/water extracts were analyzed by GC-MS and LC-DAD-MS, respectively. Histochemical tests were performed, as well as chemical analyses of leaves from adaxial and abaxial foliar surfaces of N. megapotamica, and the stereochemistry of α-bisabolol was determined. Two different chemotypes, based on volatile compounds, were identified, distinguished by the presence of isospathulenol, α-bisabolol, β-bisabolene, and (E)-nerolidol for chemotype A, and bicyclogermacrene and elemicin for chemotype B. A stereochemical analysis of chemotype A extract revealed (+)-α-bisabolol enantiomer. Histochemical tests of chemotypes showed similar results and suggested the presence of essential oil in idioblasts stained with the dye NADI. The analyses of chemotype A leaves by GC-MS revealed similar compositions for abaxial and adaxial surfaces, such pattern was also observed for chemotype B. Medium and high polarity metabolites showed high chemical similarities between the chemotypes, highlighting the presence of proanthocyanidins and glycosylated flavonoids (O- and C-glycosides). Thus, N. megapotamica produced distinct volatile chemotypes with highly conserved medium to high polarity compounds. Such results suggest that phenolic derivatives have a basal physiological function, while genetic or environmental differences lead to differentiation in volatile profiles of N. megapotamica.