Direct evidence of specific localization of sesquiterpenes and marchantin A in oil body cells of Marchantia polymorpha L.

Liverworts are a rich source of a diverse array of specialized metabolites, such as terpenoids and benzenoids, which are potentially useful for pharmaceutical or agrochemical applications, and also provide clues to elucidate the strategy by which liverworts adapt to the terrestrial environment. Liverworts, belonging to orders Marchantiales and Jungermanniales, possess oil bodies. In Marchantia polymorpha L., oil bodies are confined to scattered idioblastic oil body cells. It has been assumed that the specialized metabolites in M. polymorpha specifically accumulate in the oil bodies in oil body cells; however, no direct evidence was previously available for this specific accumulation. In this study, direct evidence was obtained using micromanipulation techniques coupled with MS analysis that demonstrated the specific accumulation of sesquiterpenoids and marchantin A in the oil body cells of M. polymorpha thalli. It was also observed that the number of oil body cells increased in thalli grown in low-mineral conditions. The amounts of sesquiterpenoids and marchantin A detected in crude extract prepared from the whole thallus were roughly proportional to the number of oil body cells found in a given volume of thallus, suggesting that oil body cell differentiation and sesquiterpenoid and marchantin A biosynthetic pathways are coordinated with each other.

Bioactivity of natural O-prenylated phenylpropenes from Illicium anisatum leaves and their derivatives against spider mites and fungal pathogens.

A variety of volatile phenylpropenes, C6-C3 compounds are widely distributed in the plant kingdom, whereas prenylated phenylpropenes are limited to a few plant species. In this study, we analysed the volatile profiles from Illicium anisatum leaves and identified two O-prenylated phenylpropenes, 4-allyl-2-methoxy-1-[(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyleugenol (9)] and 5-allyl-1,3-dimethoxy-2-(3-methylbut-2-en-1-yl)oxy]benzene [O-dimethylallyl-6-methoxyeugenol (11)] as major constituents. The structure-activity relationship of a series of eugenol derivatives showed that specific phenylpropenes, including eugenol (1), isoeugenol (2) and 6-methoxyeugenol (6), with a phenolic hydroxy group had antifungal activity for a fungal pathogen, whereas guaiacol, a simple phenolic compound, and allylbenzene had no such activity. The eugenol derivatives that exhibited antifungal activity, in turn, had no significant toxicant property for mite oviposition. Interestingly, O-dimethylallyleugenol (9) in which the phenolic oxygen was masked with a dimethylallyl group exhibited a specific, potent oviposition deterrent activity for mites. The sharp contrast in structural requirements of phenylpropenes suggested distinct mechanisms underlying the two biological activities and the importance of a phenolic hydroxy group and its dimethylallylation for the structure-based design of new functional properties of phenylpropenes.

Production of prenylated flavonoids in tomato fruits expressing a prenyltransferase gene from Streptomyces coelicolor A3(2).

Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.

Molecular characterization of fatty acid alpha-hydroperoxide-forming enzyme (alpha-oxygenase) in rice plants.

Genes encoding an alpha-oxygenase, in Nicotiana tabacum and Arabidopsis thaliana, have been recently isolated. However, the reaction mechanism of the enzyme has not so far been elucidated. In this study, a cDNA encoding the fatty acid alpha-oxygenase gene in rice plants was isolated. The deduced amino acid sequence showed high similarity (63.6%) to that of N. tabacum. The gene was cloned into an expression vector system, pQE-30, and expressed in Escherichia coli as a host cell. Palmitic acid as a substrate was incubated with the lysate of the cells, and the products were analysed by HPLC. A compound formed predominantly by the recombinant enzyme was shown to be n-pentadecanal. By incubating the mixture at 0 degrees C, 2-hydroperoxypalmitic acid was detected as a primary product and little formation of n-pentadecanal was detected. Furthermore, uptake of molecular oxygen was observed with an oxygen electrode. This indicated that the gene in rice plants encodes the alpha-oxygenase.