shikonin and ethylene

The phytohormone ethylene (ET) is a crucial signaling molecule that induces the biosynthesis of shikonin and its derivatives in Lithospermum erythrorhizon shoot cultures. However, the molecular mechanism and the positive regulators involved in this physiological process are largely unknown. In this study, the function of LeACS-1, a key gene encoding the 1-aminocyclopropane-1-carboxylic acid synthase for ET biosynthesis in L. erythrorhizon hairy roots, was characterized by using overexpression and RNA interference (RNAi) strategies. The results showed that overexpression of LeACS-1 significantly increased endogenous ET concentration and shikonin production, consistent with the up-regulated genes involved in ET biosynthesis and transduction, as well as the genes related to shikonin biosynthesis. Conversely, RNAi of LeACS-1 effectively decreased endogenous ET concentration and shikonin production and down-regulated the expression level of above genes. Correlation analysis showed a significant positive linear relationship between ET concentration and shikonin production. All these results suggest that LeACS-1 acts as a positive regulator of ethylene-induced shikonin biosynthesis in L. erythrorhizon hairy roots. Our work not only gives new insights into the understanding of the relationship between ET and shikonin biosynthesis, but also provides an efficient genetic engineering target gene for secondary metabolite production in non-model plant L. erythrorhizon.

Topics: Cloning, Molecular; Computational Biology; DNA, Complementary; Ethylenes; Gene Expression Regulation, Plant; Lithospermum; Lyases; Naphthoquinones; Plant Proteins; Plant Roots; Plants, Genetically Modified; Signal Transduction

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask(-1)) was observed when PFD-aerated has been used and it resulted in about 50% higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system).

Topics: Biomass; Boraginaceae; Chromatography, High Pressure Liquid; Culture Media; Drugs, Chinese Herbal; Ethylenes; Fluorocarbons; Naphthoquinones; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques

Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.

Topics: Boraginaceae; Cells, Cultured; DNA, Complementary; Electrophoresis, Agar Gel; Ethylenes; Expressed Sequence Tags; Gene Expression Profiling; Gene Expression Regulation, Plant; Gene Library; Genes, Plant; Light; Naphthoquinones; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Sequence Analysis, DNA; Sequence Homology, Amino Acid

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.

Topics: Amino Acids, Cyclic; Ethylenes; Glycine; Lithospermum; Naphthoquinones; Plant Shoots; Silver