SNARE-RNAi Results in Higher Terpene Emission from Ectopically Expressed Caryophyllene Synthase in Nicotiana benthamiana

Plants produce numerous terpenes and much effort has been dedicated to the identification and charac- terization of the terpene biosynthetic genes. However, little is known about how terpenes are transported within the cell and from the cell into the apoplast. To investigate a putative role of vesicle fusion in this pro- cess, we used Agrobacterium tumefaciens-mediated transient coexpression in Nicotiana benthamiana of an MtVAMP721e-RNAi construct （Vi） with either a caryophyllene synthase or a linalool synthase, respec- tively. Headspace analysis of the leaves showed that caryophyllene or linalool emission increased about five-fold when N. benthamiana VAMP72 function was blocked. RNA sequencing and protein ubiquitination analysis of the agroinflltrated N. benthamiana leaf extracts suggested that increased terpene emissions may be attributed to proteasome malfunction based on three observations： leaves with TPS＋Vi showed （1） a higher level of a DsRed marker protein, （2） a higher level of ubiquitinated proteins, and （3） coordinated induced expression of multiple proteasome genes, presumably caused by the lack of proteasome- mediated feedback regulation. However, caryophyllene or linalool did not inhibit proteasome-related pro- tease activity in the in vitro assays. While the results are not conclusive for a role of vesicle fusion in terpene transport, they do show a strong interaction between inhibition of vesicle fusion and ectopic expression of certain terpenes. The results have potential applications in metabolic engineering.