Elucidation of the biochemical pathway of 2-phenylethanol from shikimic acid using isolated protoplasts of rose flowers

Abstract

The isolated protoplasts of rose flowers were used to investigate the metabolic pathway in rose flower leading from shikimic acid or L-phenylalanine (L-Phe) to 2-phenylethanol (2PE), a dominant volatile compound in hybrid roses such as Rosa damascena Mill., R. 'Hoh-Jun', and R. 'Yves Piaget'. Deuterium-labeled L-Phe ([2H8]L-Phe) was supplied to the protoplasts isolated from R. 'Yves Piaget' petals. The volatile end products ([2Hn]-2PE, n=6-8) and their related intermediates ([2Hn]phenylacetaldehyde, n=6-8) were detected in the protoplasts by gas chromatography-mass spectrometry (GC-MS). In addition, we chemically synthesized [2,3,4,5,6-13C5]shikimic acid, a new stable isotopomer, to investigate the formation of 2PE from shikimic acid by GC-MS and nuclear magnetic resonance. We proposed the hypothetical biochemical pathway of 2PE from shikimic acid via chorismic acid, L-Phe, and phenylacetaldehyde. This protoplast system facilitates findings of metabolic intermediates and simplifies the complex branching biosynthetic pathways of floral scents to distinct individual events.

Identification of rose phenylacetaldehyde synthase by functional complementation in yeast

“…In contrast, in petunia, a gene belonging to L-tyrosine/3,4-dihydroxy-L-phenylalanine (L-Dopa) decarboxylase codes for the bifunctional enzyme phenylacetaldehyde synthase (PhPAAS), which catalyzes decarboxylation and oxidative deamination of Phe to PAA (Kaminaga et al 2006). Feeding of rose flowers with deuterium-labeled Phe implicated phenylpyruvate or 2-phenylethylamine as intermediates (Boatright et al 2004;Hayashi et al 2004;Watanabe et al 2002;Yang et al 2009) in PEA production, but PAAS-like enzymatic activity has also been recently reported (Sakai et al 2007). In most plants, PAA is rapidly metabolized to PEA by the action of a reductase/dehydrogenase and in tomato, two genes coding for phenylacetaldehyde reductases have recently been identified and characterized (Sakai et al 2007;Tieman et al 2007).…”

Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts

“…Additionally, we encountered the limitations of detecting the intermediates of metabolic pathways due to the dilution of isotope-labeled compounds with endogenous metabolites (Sayama, 2008). Based on the above considerations, we previously developed a simple and controllable approach to elucidate the biosynthesis of 2PE in rose using isolated rose petal protoplasts and confirmed the incorporation of 13 C-labeled shikimic acid into 2PE (Yang et al, 2009). Although isolated protoplasts are an artificial system, this model should reveal some fundamental information regarding the biogenesis of 2PE due to the higher conversion rate of exogenously applied precursors within a short incubation period.…”

“…The stages of floral growth and the preparation of protoplasts have been described previously (Hayashi et al, 2004;Yang et al, 2009). l-Phe and l-[ 2 H 8 ]Phe (2.5 mol) were dissolved in protoplast buffer and added to the protoplasts.…”

“…The volatile compounds were analyzed as described previously (Yang et al, 2009). The details conditions are described in the 'Supplementary information'.…”

“…2-Phenylethanol (2PE) is one of the prominent scent compounds produced by Damask roses (Hayashi et al, 2004;Sakai et al, 2007;Yang et al, 2009), and in various fruits such as strawberry, tomato and grape varieties (Aubert et al, 2005). 2PE and phenylacetaldehyde (PAld) contribute toward characteristic flavors in wine and cheese (Marilley and Casey, 2004) producing a pleasantly sweet, flowery note at low concentrations, while PAld is nauseating and Abbreviations: 2PE, 2-phenylethanol; AAAT, aromatic amino acid aminotransferase; AADC, aromatic amino acid decarboxylase; AspAT, aspartate aminotransferase; AlaAT, alanine aminotransferase; E. coli, Escherichia coli; PAld, phenylacetaldehyde; PAR, phenylacetaldehyde reductase; l-Phe, l-phenylalanine; PheAT, phenylalanine aminotransferase; PLP, pyridoxal-5 -phosphate; PPA, phenylpyruvic acid; TrpAT, tryptophan aminotransferase; TyrAT, tyrosine aminotransferase.…”