Heterologous Expression of Methylketone Synthase1 and Methylketone Synthase2 Leads to Production of Methylketones and Myristic Acid in Transgenic Plants

Geng Yu,Eran Pichersky

doi:10.1104/pp.113.228502

Abstract

Some plants produce methylketones as potent defense compounds against various insects. Wild tomato (Solanum habrochaites), a relative of the cultivated tomato (Solanum lycopersicum), synthesizes large amounts of 2-methylketones in its glandular trichomes, but cultivated tomato trichomes contain little or no methylketones. Two enzymes, Solanum habrochaites methylketone synthase1 (ShMKS1) and ShMKS2, are required to convert β-ketoacyl acyl-carrier protein intermediates of the fatty acid biosynthetic pathway to methylketones. ShMKS2 is a thioesterase that hydrolyzes β-ketoacyl acyl-carrier protein, and ShMKS1 is a decarboxylase that converts the resulting 3-ketoacids to 2-methylketones. We introduced ShMKS2 by itself or together with ShMKS1 to Arabidopsis (Arabidopsis thaliana), tobacco (Nicotiana tabacum), and cultivated tomato under the control of the 35S, Rubisco small subunit, and tomato trichome-specific promoters. Young tobacco and Arabidopsis plants expressing both genes under the control of 35S and Rubisco small subunit promoters produced methylketones in their leaves but had serious growth defects. As plants matured, they ceased to produce methylketones. Tobacco plants but not Arabidopsis or tomato plants expressing only ShMKS2 under the 35S promoter also synthesized methylketones, but at a lower rate. Transgenic cultivated tomato plants expressing ShMKS1 and ShMKS2 under trichome-specific promoters had slightly elevated levels of methylketone. Trace amounts of myristic acid were also detected in transgenic plants constitutively expressing ShMKS2 with or without ShMKS1. These results suggest that increases in methylketone production in plants will require the targeting of the pathway to self-contained structures in the plant and may also require increasing the flux of fatty acid biosynthesis.

Highlights

Some plants produce methylketones as potent defense compounds against various insects
To generate transgenic Arabidopsis lines expressing both Solanum habrochaites methylketone synthase1 (ShMKS1) and ShMKS2, the open reading frame (ORF) of ShMKS1 was fused to a Rubisco small subunit promoter and added to the p35S::ShMKS2 expression vector
The presence of intact ORF sequences of ShMKS1 and/or ShMKS2 was verified by genomic DNA PCR

Summary

Introduction

Some plants produce methylketones as potent defense compounds against various insects. Young tobacco and Arabidopsis plants expressing both genes under the control of 35S and Rubisco small subunit promoters produced methylketones in their leaves but had serious growth defects. As plants matured, they ceased to produce methylketones. Trace amounts of myristic acid were detected in transgenic plants constitutively expressing ShMKS2 with or without ShMKS1 These results suggest that increases in methylketone production in plants will require the targeting of the pathway to self-contained structures in the plant and may require increasing the flux of fatty acid biosynthesis. Transgenic Plants Expressing Methylketone Synthase and MKS2 methylketone-producing line, followed by gene expression comparison with a trichome EST database from wild tomato Additional genetic and genomic analyses identified a second gene, designated ShMKS2, whose expression was positively associated with methylketone production (BenIsrael et al, 2009)

Methods

Results

Conclusion