title: Functional characterisation of genes involved in pyridine alkaloid biosynthesis in tobacco.
abstract: Although secondary metabolism in Nicotiana tabacum (L.) (tobacco) is rather well studied, many molecular aspects of the biosynthetic pathways and their regulation remain to be disclosed, even for prominent compounds such as nicotine and other pyridine alkaloids. To identify players in tobacco pyridine alkaloid biosynthesis a functional screen was performed, starting from a tobacco gene collection established previously by means of combined transcript profiling and metabolite analysis. First, full-length cDNA clones were isolated for 34 genes, corresponding to tobacco transcript tag sequences putatively associated with pyridine alkaloid metabolism. Full-length open reading frames were transferred to pCaMV35S-steered overexpression vectors. The effects of plant transformation with these expression cassettes on the accumulation of nicotine and other pyridine alkaloids were assessed in transgenic tobacco Bright-Yellow 2 (BY-2) cell suspensions and hairy root cultures. This screen identified potential catalysers of tobacco pyridine metabolism, amongst which a lysine decarboxylase-like gene and a GH3-like enzyme. Overexpression of the GH3-like enzyme, presumably involved in auxin homeostasis and designated NtNEG1 (Nicotiana tabacum Nicotine-Enhancing GH3 enzyme 1), increased nicotine levels in BY-2 hairy roots significantly. This study shows how functional genomics-based identification of genes potentially involved in biosynthetic pathways followed by systematic functional assays in plant cells can be used at large-scale to decipher plant metabolic networks at the molecular level.
authors.affiliation: VTT Technical Research Centre of Finland, P.O. Box 1000, FIN-02044 VTT (Espoo), Finland.
authors.forename: Suvi T
authors.initials: ST
authors.lastname: Häkkinen
authors.forename: Sofie
authors.initials: S
authors.lastname: Tilleman
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Swiatek
authors.forename: Valerie
authors.initials: V
authors.lastname: De Sutter
authors.forename: Heiko
authors.initials: H
authors.lastname: Rischer
authors.forename: Isabelle
authors.initials: I
authors.lastname: Vanhoutte
authors.forename: Harry
authors.initials: H
authors.lastname: Van Onckelen
authors.forename: Pierre
authors.initials: P
authors.lastname: Hilson
authors.forename: Dirk
authors.initials: D
authors.lastname: Inzé
authors.forename: Kirsi-Marja
authors.initials: KM
authors.lastname: Oksman-Caldentey
authors.forename: Alain
authors.initials: A
authors.lastname: Goossens
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry; D002460:Cell Line; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000737:chemistry / Q000378:metabolism; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000470:Alkaloids; D010940:Plant Proteins; D011725:Pyridines; C023666:pyridine