Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

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 about 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 advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

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.