Production mechanism of β-thujaplicin by cell cultures of woody species

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β-Thujaplicin, called as hinokitiol in Japanese, is one of plant tropolones, contributing to the durability of heartwood of many Cupressaceae species due to its broad antifungal and antibacterial spectra. We have studied the mechanism of the production of β-thujaplicin by cell cultures of Cupressus lusitanica, a species in the Curpressaceae family.At first, a signal transduction pathway from elicitor receptor(s) on the cell surface to the onset of β-thujaplicin biosynthesis was investigated. It was elucidated by accelerator and/or inhibitor feeding experiments that G-protein, lipoxygenase, and protein kinases are involved in the signal transduction, and cAMP and Ca cation functioned as messengers. The jasmonate pathway is suggested to play an important role in the transduction system leading to β-thujaplicin production.Terpinolene synthase was found to be the most probable enzyme that is involved in biosynthesis of an intermediate monoterpene, most probably the direct precursor of β-thujaplicin. For cloning the gene of this enzyme by means of RT-PCR and cDNA screening, we obtained three DNA clones that may code the terpinolene synthase from a cDNA library of an elicitor-treated cell culture of C.lusitanica, because it was found that the enzyme was activated by the action of the elicitor. Then, the gene(s) that is involved in the β-thujaplicin biosynthesis was searched by the micro array method, for it was expected that mRNA(s) induce by the elicitation should reflect the β-thujaplicin biosynthesis gene(s). In cDNA library of C.lusitanica tells, it was observed that 48 clones exhibited significant differences in the strength between before and after the elicitor treatment. Function of these clones should be studied in future works.