Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. the biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (ViGS) systems, which have been shown advantageous in introducing iRnA genes into non-model plants. this study describes the development of a ViGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. the levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRnA was detected in infected plants but not in escape and mock plants. Quantitative pcR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the ViGS system silence target genes in soil-grown L. erythrorhizon. Abbreviations qRT-PCR Quantitative reverse-transcriptase mediated polymerase chain reaction VIGS Virus-induced gene silencing PDS Phytoene desaturase ALSV Apple latent spherical virus ACT Actin UTR Untranslated region CMV Cucumber mosaic virus Lithospermum erythrorhizon is a Boraginaceaeous medicinal plant that produces a unique red naphthoquinone, shikonin, which accumulates exclusively in its roots. The dried roots have been used as a crude drug in Asian countries, with shikonin derivatives being major active pharmaceutical components of these herbal medicines. Shikonin and its stereo-isomer alkannin have been reported to have various biological activities, including antibacterial, anti-inflammatory, anti-oxidant, antitumor, anti-angiogenic 1 , and anti-topoisomerase 2 activities. These compounds have also been found to enhance granulation and glucose uptake 3 and to reduce adiposity 4. These natural pigments are also used as dyes, especially for cloth, and to have other commercial uses. Although

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites taking its advantages, such as the high production rate and the clear inducibility using culture conditions. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plant. Microscopic analysis showed that shikonin is accumulated in a vast number of particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24 to 38% of total TAG), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that TAG encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for TAG as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. The mechanism of secretion remains largely unknown, because hydrophobic metabolites, which may form oil droplets or crystals in cytosol, inducing cell death, cannot be directly secreted by transporters. Moreover, some secondary metabolic lipids react with cytosolic components leading to their decomposition. Lipophilic metabolites should thus be solubilized by matrix lipids and compartmentalized by membrane lipids. The mechanism of lipophilic metabolite secretion was assessed using shikonin, a red naphthoquinone lipid, in Lithospermum erythrorhizon. Cell secretion of shikonin also involved the secretion of about 30% of triacylglycerol (TAG), composed predominantly of saturated fatty acids. Shikonin production was associated with the induction of large amounts of the membrane lipid phosphatidylcholine. Together with in vitro reconstitution, these findings suggest a novel role for TAG as a matrix lipid for the secretion of lipophilic metabolites.