13C NMR analysis of polyphenol biosynthesis in grape cells: Impact of various inducing factors

Abstract

13C NMR spectroscopy was used as a complement to high performance liquid chromatography (HPLC) or spectrophotometry to analyse stilbene and anthocyanin metabolism in grape cell cultures. Stilbene and anthocyanin biosynthesis are closely related. Anthocyanins are major components of grape quality whereas stilbenes play a crucial role in defence mechanisms and are strongly correlated to natural grapevine resistance. Stimulation of stilbene production by natural inducers offers an interesting alternative to the use of pesticides in plant protection strategies. NMR allowed us to analyse both stilbene and anthocyanin pathways by following step by step the incorporation of the labelled precursor [1- 13C] l-phenylalanine along the entire biosynthetic routes. However, several intermediates of the anthocyanin biosynthetic pathways remained unobservable, providing evidence for metabolic channelling. The effect of various elicitors on stilbene and anthocyanin biosynthesis was also investigated. In complement to quantification of the end-products by HPLC and spectrophotometry, 13C NMR studies provided information at the biosynthetic level. Sucrose addition stimulated the biosynthesis of anthocyanins without influencing stilbenes. Methyljasmonate and fungal elicitor strongly increased stilbene production through the activation of enzymes from phenylalanine ammonia-lyase to stilbene synthase. However, methyljasmonate showed an inhibitory effect on anthocyanin biosynthesis, suggesting the existence of a competition between the two pathways. 13C NMR spectroscopy, combined with analytical techniques such as HPLC and spectrophotometry, provides a very interesting tool to better understand the mechanisms underlying the production of secondary metabolites and the metabolic processes of plant resistance induction by elicitation.