title: SNARE-RNAi results in higher terpene emission from ectopically expressed caryophyllene synthase in Nicotiana benthamiana.
abstract: Plants produce numerous terpenes and much effort has been dedicated to the identification and characterization 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 process, 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, respectively. 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 agroinfiltrated 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 protease 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.
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, PO Box 658, 6700 AR Wageningen, The Netherlands.
authors.forename: Hieng-Ming
authors.initials: HM
authors.lastname: Ting
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, PO Box 658, 6700 AR Wageningen, The Netherlands.
authors.forename: Thierry L
authors.initials: TL
authors.lastname: Delatte
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, PO Box 658, 6700 AR Wageningen, The Netherlands.
authors.forename: Pim
authors.initials: P
authors.lastname: Kolkman
authors.affiliation: Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany.
authors.forename: Johana C
authors.initials: JC
authors.lastname: Misas-Villamil
authors.affiliation: Plant Chemetics Laboratory, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany; Plant Chemetics Laboratory, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.
authors.forename: Renier A L
authors.initials: RA
authors.lastname: van der Hoorn
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, PO Box 658, 6700 AR Wageningen, The Netherlands.
authors.forename: Harro J
authors.initials: HJ
authors.lastname: Bouwmeester
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, PO Box 658, 6700 AR Wageningen, The Netherlands. Electronic address: sander.vanderkrol@wur.nl.
authors.forename: Alexander R
authors.initials: AR
authors.lastname: van der Krol
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D046913:Medicago truncatula / Q000235:genetics; D060847:Metabolic Engineering; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D000081223:Polycyclic Sesquiterpenes; D034622:RNA Interference; D050600:SNARE Proteins / Q000235:genetics* / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000081223:Polycyclic Sesquiterpenes; D050600:SNARE Proteins; D012717:Sesquiterpenes; C024714:caryophyllene; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase
keywords: Nicotiana benthamiana; caryophyllene synthase; linalool synthase; proteasome; terpene transport; vesicle-associated membrane proteins (VAMP72)