A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon

Lithospermum erythrorhizon produces red naphthoquinone pigments that are sh ikonin derivatives. They are accumulated exclusively in the roots of this p lant. The biosynthesis of shikonin is strongly inhibited by light, even tho ugh other environmental conditions are optimized. Thus, L. erythuorhizon da rk-inducible genes (LeDIs) were isolated to investigate the regulatory mech anism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific ex pression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a simila r pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydropho bic polypeptide of 114 amino acids that shared significant similarities wit h some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Red uction of LeDI-2 expression by its antisense DNA in hairy roots of L. eryth rorhizon decreased the shikonin accumulation, whereas other biosynthetic en zymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a cri tical biosynthetic step, showed similar activity as the wild-type clone. Th is is the first report of the gene that is involved in production of second ary metabolites without affecting biosynthetic enzyme activities.