Two 4-coumarate: Coenzyme A ligase genes involved in acteoside and flavonoids biosynthesis in Rehmannia glutinosa

Abstract

Acteoside and flavonoids as important natural products have pharmacological activities in Rehmannia glutinosa Libosch., which is of great economic significance for leading to efforts to enhance the productions. Coumarate: coenzyme A (CoA) ligases (4CLs) in higher plants could play pivotal roles in branching points at which the metabolic fluxes are directed to either the acteoside or flavonoids biosynthetic pathways. To investigate the molecular regulation in acteoside and flavonoids biosynthesis, the study identified and characterized two Rehmannia glutinosa 4CL genes (Rg4CL1 and Rg4CL2) by in silico and experimental analysis, revealing a 53.35–81.68 % protein sequence identity with equivalents from other plants, two highly conserved sequence motifs and subcellular localization to the cytosol. Phylogenetic and enzyme kinetic analyses indicated that Rg4CL1 belonged to Class I of this enzyme family, and was the highest activity towards caffeic acid, while Rg4CL2 grouped with Class II and efficiently catalyzed p-coumaric acid and cinnamic acid. The overexpression of Rg4CL1 and Rg4CL2 in Rehmannia glutinosa significantly increased acteoside and flavonoids productions, especially the acteoside accumulation from the Rg4CL1 overexpression lines and flavonoids accumulation from the Rg4CL2 overexpression lines. The results revealed that the Rg4CLs are involved in acteoside and flavonoids biosynthesis, especially, Rg4CL1 and Rg4CL2 may preferentially be responsible for the flux diversions towards acteoside and flavonoids biosynthesis, respectively. The study is the first to elucidate the molecular function of the Rg4CL isoforms in the biosynthetic pathways of acteoside and flavonoids, providing insights into the increase of natural active products from the phenylpropanoid pathway in Rehmannia glutinosa.