Lithospermum erythrorhizon cell cultures: Present and future aspects

Abstract

Lithospermum erythrorhizon cell cultures have been used to produce plant secondary metabolites, as well as in biosynthetic studies. Shikonin, a representative secondary metabolite of L. erythrorhizon, was first produced industrially by dedifferentiated cell cultures in the 1980s. This culture system has since been used in research on various plant secondary metabolites. Other boraginaceaeous plant species, including Arnebia, Echium, Onosma and Alkanna, have been shown to produce shikonin, and studies have assessed shikonin regulation, including transgene expression, in these plants. This review summarizes current knowledge of shikonin production by L. erythrorhizon cell and hairy root cultures, including the historical aspect of large-scale production, and discusses future biochemical and biological research using this species.

Keywords: Lithospermum erythrorhizon; lithospermic acid B; plant cell cultures; shikonin derivatives.

Figure 1. Intact plant and cell suspension cultures of Lithospermum erythrorhizon. (A) An intact L. erythrorhizon plant. The Japanese name ‘Murasaki’ is the representative name of purple color in Japanese. Flowers are white, whereas the roots are dark red to dark purple in color due to the high accumulation of shikonin derivatives in the outer bark and cork layer of the roots. The dried roots are used as a crude drug in Japanese and Chinese traditional medicines. (B) Close up of flowers. (C) Intact root of a young L. erythrorhizon plant. (D) L. erythrorhizon cells cultured in M9 medium (left) producing shikonin derivatives, and in LS medium (right), used for cell subculture.

Figure 2. Scheme of large-scale culture set-up for production of shikonin. L. erythrorhizon cells were cultured for 9 days in MG5 medium, a modified LS medium for high cell growth. The MG5 medium was removed by filtration, and L. erythrorhizon cells were transferred to a production tank and cultured for 14 days in M9 medium. Shikonin derivatives mainly attached to the cell surface and were easily extracted from the filtered cell mass after drying.

Figure 3. Secondary metabolism in cultured L. erythrorhizon cells. Shikonin exists in living plant cells as esters of low molecular weight fatty acids, such as acetate, whereas free shikonin is undetectable. Inhibition of shikonin production in LS medium results in the accumulation in vacuoles of the aromatic intermediate, p-hydroxybenzoic acid (PHB), as its O-glucoside. Induction of PHB geranyltransferase and inhibition of naphthalene ring formation results in the accumulation of large amounts of dihydroechinofuran, partially oxidized to the orange compound, echinofuran B, in both media. In addition to secreting these quinone metabolites, L. erythrorhizon cells produce large amounts of the phenylpropanoid tetramer, lithospermic acid B, which accumulates inside the cells. End products that show high accumulation are highlighted with squares.

Figure 4. Schematic model of shikonin secretion from L. erythrorhizon cells. Electron microscopic studies suggested that the shikonin-producing cell contained electron-dense vesicles derived from endoplasmic reticulum. These vesicles may cross the plasma membrane for excretion by unknown mechanisms, with shikonin derivatives accumulating as red granules on cell walls. Many small particles were found beneath the shikonin-rich red granules attached to the cell surface. Because shikonin derivatives are highly hydrophobic, these intracellular vesicles are regarded as lipid monolayers, similar to oil bodies (Tatsumi et al. 2016).

Figure 5. Hairy roots of L. erythrorhizon. (A) Hairy root cultures in M9 medium under illumination (left) and in the dark (right). (B, C) Longitudinal sections of a hairy root grown under illumination (B) and in the dark (C). Shikonin derivatives accumulatd in epidermal cells. (D) Whole mount picture of a GUS transformant of an L. erythrorhizon hairy root. Scale bars are 20 µm.