Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures

When cells of Lithospermum erythrorhizon were transferred from B5 solid medium into M9 liquid medium and grown in the dark, they produced and accumulated shikonin and its derivatives; meanwhile, transcripts of PAL, 4CL, HMGR, PGT, and CYP98A6 were rapidly induced within 2 h, peaking within 6 h, and then decreasing over time. However, when L. erythrorhizon cells were cultured in M9 medium and grown under white light, a negative regulator for the biosynthesis of shikonin and its derivatives, the stimulating effect of the medium transition on the transcription of these genes was also displayed with similar patterns over 24 h of culture period. To investigate the time-dependent effect of light signals on regulating gene transcription, expression analysis of L. erythrorhizon dark-inducible genes LeDI-1-LeDI-5 was conducted. No obvious inhibitory effects of light signals on the transcription of LeDI-1-LeDI-5 were detected during the first 12 h, but these genes, in particular LeDI-2 and LeDI-4, were suppressed by light signals to some degree during longer culture periods, 12 h to 10 days. These findings suggest that light signals are likely to inhibit the transcription of the shikonin-biosynthetic genes indirectly or under long-term conditions.