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-methoxyindol-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.

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…The four CYP81Fs have partly overlapping product specificity: CYP81F1, CYP81F2 and CYP81F3 catalyse hydroxylation of I3M to 4OH-I3M while all four CYP81Fs catalyse the conversion of I3M to 1-OH-I3M, when tested in the transient expression system in Nicotiana benthamiana. Mutant analysis partly confirms the biochemical analysis and, combined with expression analysis based on DNA microarrays, suggests that the CYP81Fs are expressed in an organ-or tissue-specific manner, thus representing both neo-and subfunctionalization [111].…”

“…Arabidopsis contains four different indole glucosinolates derived from tryptophan. The core indole glucosinolates indol-3-yl-methyl (I3M) is hydroxylated and methoxylated by a set of P450s and methyl-transferases [111]. There are four CYP82s in Arabidopsis: CYP81F3 and CYP81F4 are situated in tandem, spaced from CYP81F1 by genes with unknown function, on chromosome 4, whereas CYP81F2 is positioned on chromosome five.…”

Indole Glucosinolate Biosynthesis Limits Phenylpropanoid Accumulation in Arabidopsis thaliana

“…The synthesis of indole glucosinolates starts with the conversion of tryptophan to indole-3-acetaldoxime (IAOx) by two cytochrome P450-dependent monooxygenases (P450s), CYP79B2 and CYP79B3 (Hull et al, 2000;Mikkelsen et al, 2000Mikkelsen et al, , 2004. Subsequently CYP83B1 catalyzes the conversion of IAOx into 1-aci-nitro-2-indolyl-ethane and then multiple further modifications result in the production of the various indole glucosinolates (Pfalz et al, 2009(Pfalz et al, , 2011. IAOx is also a precursor for indole-3-acetic acid (IAA), a phytohormone, and camalexin, an important phytoalexin in Arabidopsis (Glawischnig et al, 2004;Nafisi et al, 2007).…”

Biosynthesis of cabbage phytoalexins from indole glucosinolate

“…Because tobacco does not natively synthesize glucosinolates, we simultaneously expressed eight genes from B. rapa, predicted to comprise the indole glucosinolate pathway (IG pathway) based on orthology to characterized enzymes in Arabidopsis (28,29). This set of genes includes the SUR1 ortholog that we characterized in vitro.…”