Parallel evolution of UbiA superfamily proteins into aromatic O-prenyltransferases in plants.

Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.

Highly efficient method of Lithospermum erythrorhizon transformation using domestic Rhizobium rhizogenes strain A13.

Lithospermum erythrorhizon, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact L. erythrorhizon plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in L. erythrorhizon cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for L. erythrorhizon transformation using hairy roots by Rhizobium rhizogenes strain A13, present domestically in Japan. Hairy roots of L. erythrorhizon were generated from explants of the axenic shoots that were infected with R. rhizogenes strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50-70%. Although methods for the systematic stable transformation of L. erythrorhizon plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of L. erythrorhizon genes.