Nucleotide sequence of a cDNA from Lithospermum erythrorhizon homologous to PR-1 of parsley.

Cell-suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) grown in the dark produce a large amount of red naphthoquinone pigments that are shikonin derivatives when transferred to the production medium M9 (Fujita et al., 1981) from the Linsmaier Skoog liquid medium (Linsmaier and Skoog, 1965). It is known that shikonin biosynthesis is completely inhibited by irradiation with either white or blue light (Heide et al., 1989). In most plant cell cultures, secondary metabolism, in particular the production of phenolic compounds, is stimulated by light, and genes related to light-inducible biosynthesis of secondary metabolites have recently been investigated to identify cis-acting elements in their promoter regions and to elucidate the function of trans-acting factors (Feldbriigge et al., 1994). However, the molecular mechanism of the induction of secondary metabolism in the dark is an open question. The Lithospermum cell culture seems to offer an ideal system to study dark-inducible genes, since the shikonin production is specifically inhibited by light, whereas it can be induced rapidly when the cultures are placed in darkness. In the present study, an attempt was made to clone cDNAs expressed dominantly in the dark-grown Lithospermum cell cultures. Two different cDNA samples, one corresponding to mRNA from Lithospermum cells cultured for 2 d after inoculation in M9 medium in the dark, and the other corresponding to that from the cells cultured under continuous illumination, were prepared. cDNAs expressed preferably in the dark were enriched by the cDNA subtraction method of Wang and Brown (1991). A mixture of the subtracted cDNA was used as a probe for plaque hybridization, to detect about 360 positive signals among 6000 plaques of the A-ZAPII cDNA library prepared from Lithospermum cells cultured in M9 medium in the dark. Of the positive clones 160 single plaques were randomly picked, and a dot blot filter containing each A clone was prepared. Since about half of the A clones were found to be identical in a crosshybridization experiment with the dot blot filter, one of these plaques was subjected to further analysis.