title: Metabolic engineering in Nicotiana benthamiana reveals key enzyme functions in Arabidopsis indole glucosinolate modification.
abstract: Indole glucosinolates, derived from the amino acid Trp, are plant secondary metabolites that mediate numerous biological interactions between cruciferous plants and their natural enemies, such as herbivorous insects, pathogens, and other pests. While the genes and enzymes involved in the Arabidopsis thaliana core biosynthetic pathway, leading to indol-3-yl-methyl glucosinolate (I3M), have been identified and characterized, the genes and gene products responsible for modification reactions of the indole ring are largely unknown. Here, we combine the analysis of Arabidopsis mutant lines with a bioengineering approach to clarify which genes are involved in the remaining biosynthetic steps in indole glucosinolate modification. We engineered the indole glucosinolate biosynthesis pathway into Nicotiana benthamiana, showing that it is possible to produce indole glucosinolates in a noncruciferous plant. Building upon this setup, we demonstrate that all members of a small gene subfamily of cytochrome P450 monooxygenases, CYP81Fs, are capable of carrying out hydroxylation reactions of the glucosinolate indole ring, leading from I3M to 4-hydroxy-indol-3-yl-methyl and/or 1-hydroxy-indol-3-yl-methyl glucosinolate intermediates, and that these hydroxy intermediates are converted to 4-methoxy-indol-3-yl-methyl and 1-methoxy-indol-3-yl-methyl glucosinolates by either of two family 2 O-methyltransferases, termed indole glucosinolate methyltransferase 1 (IGMT1) and IGMT2.
authors.affiliation: Max Planck Institute for Chemical Ecology, D-07745 Jena, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Pfalz
authors.forename: Michael Dalgaard
authors.initials: MD
authors.lastname: Mikkelsen
authors.forename: Pawel
authors.initials: P
authors.lastname: Bednarek
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.forename: Barbara Ann
authors.initials: BA
authors.lastname: Halkier
authors.forename: Juergen
authors.initials: J
authors.lastname: Kroymann
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005961:Glucosinolates / Q000096:biosynthesis*; D007211:Indoles / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009154:Mutation; D018411:Spodoptera / Q000166:cytology; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D018744:DNA, Plant; D005961:Glucosinolates; D007211:Indoles; C036483:T-DNA; D003577:Cytochrome P-450 Enzyme System