Detection and Quantification of Engineered Proanthocyanidins in Transgenic Plants

Abstract

Proanthocyanidins (PAs) are oligomeric plant natural products mostly derived from epicatechin and/or catechin monomers. In studies aimed at engineering PAs into plant tissues that do not naturally make these compounds, we have expressed PA biosynthetic and regulatory genes in tobacco, alfalfa ( Medicago sativa) and the model legume Medicago truncatula. Because engineered tannins may be produced in small quantities and it is often necessary to screen many independent plant lines, we have developed an improved, highly sensitive method to quantify and determine the composition of oligomeric PAs in plant extracts. The method involves normal-phase HPLC separation of semi-purified PAs followed by post-column reaction with the PA-specific reagent DMACA (dimethylaminocinnamaldehyde). This procedure allows for accurate and sensitive quantification of individual oligomeric PAs and, unlike currently used methods, does not require exhaustive sample preparation and clean-up. Compositional data are shown for genetically engineered PAs in tobacco and alfalfa.