Tissue culture-generated shoot-based clonal lines are being used to investigate the role of proline-linked pentose phosphate pathway in stimulating rosmarinic acid (RA). In this study is reported the stimulation of RA biosynthesis in oregano clonal line O-1 in response to proline, proline precursors (ornithine and arginine), and proline analogue (azetidine-2-carboxylate, A2C). Following exogenous treatment with proline and proline precursors in the presence or absence of proline analogue A2C, significantly enhanced RA content and concurrently higher levels of endogenous proline were observed compared to control. Analogue (A2C) treatment alone stimulated highest levels of RA without any increase in endogenous proline. Endogenous proline levels on day 30, however, were significantly higher in all proline and ornithine treatments with or without A2C but not in treatments with A2C or arginine alone. The stimulation of RA synthesis in response to proline or proline precursors with or without A2C suggested that deregulation and enhancement of proline synthesis or proline oxidation may be important for RA biosynthesis in oregano. This stimulation of RA biosynthesis provided strong clues that proline synthesis may be linked to stimulation of the pentose phosphate pathway, driving key precursor metabolites toward shikimate and phenylpropanoid pathways. RA-stimulating compounds also enhanced total phenolics and hardened stem tissues, indicating possible lignification due to polymerization of phenolic metabolites.

Keywords: Arginine; azetidine-2-carboxylate; Origanum vulgare; ornithine; phenolics; proline; rosmarinic acid; shoot culture