title: Homologous and heterologous expression of grapevine E-(β)-caryophyllene synthase (VvGwECar2).
abstract: E-(β)-caryophyllene is a sesquiterpene volatile emitted by plants and involved in many ecological interactions within and among trophic levels and it has a kairomonal activity for many insect species. In grapevine it is a key compound for host-plant recognition by the European grapevine moth, Lobesia botrana, together with other two sesquiterpenes. In grapevine E-(β)-caryophyllene synthase is coded by the VvGwECar2 gene, although complete characterization of the corresponding protein has not yet been achieved. Here we performed the characterization of the enzyme after heterologous expression in E. coli, which resulted to produce in vitro also minor amounts of the isomer α-humulene and of germacrene D. The pH optimum was estimated to be 7.8, and the Kmand Kcatvalues for farnesyl pyrophosphate were 31.4 μM and 0.19 s-1respectively. Then, we overexpressed the gene in the cytoplasm of two plant species, Arabidopsis thaliana and the native host Vitis vinifera. In Arabidopsis the enzyme changed the plant head space release, showing a higher selectivity for E-(β)-caryophyllene, but also the production of thujopsene instead of germacrene D. Overall plants increased the E-(β)-caryophyllene emission in the headspace collection by 8-fold compared to Col-0 control plants. In grapevine VvGwECar2 overexpression resulted in higher E-(β)-caryophyllene emissions, although there was no clear correlation between gene activity and sesquiterpene quantity, suggesting a key role by the plant regulation machinery.
authors: Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Università 5, 39100 Bolzano, Italy; Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: umberto.salvagnin@fmach.it.
authors: Umberto
authors: U
authors: Salvagnin
authors: Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: silvia.carlin@fmach.it.
authors: Silvia
authors: S
authors: Carlin
authors: Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Università 5, 39100 Bolzano, Italy. Electronic address: Sergio.Angeli@unibz.it.
authors: Sergio
authors: S
authors: Angeli
authors: Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: urska.vrhovsek@fmach.it.
authors: Urska
authors: U
authors: Vrhovsek
authors: Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: gianfranco.anfora@fmach.it.
authors: Gianfranco
authors: G
authors: Anfora
authors: Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: mickael.malnoy@fmach.it.
authors: Mickael
authors: M
authors: Malnoy
authors: Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, 38010 S. Michele all'Adige, TN, Italy. Electronic address: stefan.martens@fmach.it.
authors: Stefan
authors: S
authors: Martens
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D017360:Arabidopsis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D000081222:Monocyclic Sesquiterpenes; D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D027843:Vitis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D055549:Volatile Organic Compounds / Q000032:analysis
chemical_list: D000081222:Monocyclic Sesquiterpenes; D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D055549:Volatile Organic Compounds; C042686:humulene; C004808:farnesyl pyrophosphate; C024714:caryophyllene; C433908:thujopsene; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase
keywords: Arabidopsis thaliana Brassicaceae; E-(β)-Caryophyllene; Genetic transformation; Grapevine; Sesquiterpene(s); TPS; Terpene synthase; VOC(s); Vitis vinifera Vitaceae