title: Metabolic engineering of geranic acid in maize to achieve fungal resistance is compromised by novel glycosylation patterns.
abstract: Many terpenoids are known to have antifungal properties and overexpression of these compounds in crops is a potential tool in disease control. In this study, 15 different mono- and sesquiterpenoids were tested in vitro against two major pathogenic fungi of maize (Zea mays), Colletotrichum graminicola and Fusarium graminearum. Among all tested terpenoids, geranic acid showed very strong inhibitory activity against both fungi (MIC<46 μM). To evaluate the possibility of enhancing fungal resistance in maize by overexpressing geranic acid, we generated transgenic plants with the geraniol synthase gene cloned from Lippia dulcis under the control of a ubiquitin promoter. The volatile and non-volatile metabolite profiles of leaves from transgenic and control lines were compared. The headspaces collected from intact seedlings of transgenic and control plants were not significantly different, although detached leaves of transgenic plants emitted 5-fold more geranyl acetate compared to control plants. Non-targeted LC-MS profiling and LC-MS-MS identification of extracts from maize leaves revealed that the major significantly different non-volatile compounds were 2 geranic acid derivatives, a geraniol dihexose and 4 different types of hydroxyl-geranic acid-hexoses. A geranic acid glycoside was the most abundant, and identified by NMR as geranoyl-6-O-malonyl-β-d-glucopyranoside with an average concentration of 45μM. Fungal bioassays with C. graminicola and F. graminearum did not reveal an effect of these changes in secondary metabolite composition on plant resistance to either fungus. The results demonstrate that metabolic engineering of geraniol into geranic acid can rely on the existing default pathway, but branching glycosylation pathways must be controlled to achieve accumulation of the aglycones.
authors.affiliation: Plant Research International, Wageningen UR, The Netherlands.
authors.forename: Ting
authors.initials: T
authors.lastname: Yang
authors.forename: Geert
authors.initials: G
authors.lastname: Stoopen
authors.forename: Nasser
authors.initials: N
authors.lastname: Yalpani
authors.forename: Jacques
authors.initials: J
authors.lastname: Vervoort
authors.forename: Ric
authors.initials: R
authors.lastname: de Vos
authors.forename: Alessandra
authors.initials: A
authors.lastname: Voster
authors.forename: Francel W A
authors.initials: FW
authors.lastname: Verstappen
authors.forename: Harro J
authors.initials: HJ
authors.lastname: Bouwmeester
authors.forename: Maarten A
authors.initials: MA
authors.lastname: Jongsma
mesh_terms: D000080462:Acyclic Monoterpenes; D000935:Antifungal Agents / Q000378:metabolism*; D020231:Colletotrichum / Q000254:growth & development; D005670:Fusarium / Q000254:growth & development; D032411:Lippia / Q000201:enzymology / Q000235:genetics; D010935:Plant Diseases / Q000235:genetics / Q000517:prevention & control*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D030821:Plants, Genetically Modified; D013729:Terpenes / Q000378:metabolism*; D003313:Zea mays / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000382:microbiology
chemical_list: D000080462:Acyclic Monoterpenes; D000935:Antifungal Agents; D013729:Terpenes; C007836:geraniol; C043081:decaprenoic acid