| Shikonin and its derivatives are a class of naphthoquinone-containing pigments only synthesized in the roots of Boraginaceae plants, such as Lithospermum erythorhizon sieb. et zucc, Onosma paniculatum Bur. et Franch and Arnebia euchroma(Royle) Johnst. These compounds possess significant anti-inflammatory, antitumor, and antimicrobial activities. Recently, natural naphthoquinone pigments have been widely used as natural colorants to replace synthetic dyes, and shikonin and its derivatives have been increasingly used as natural colorant either for food or pharmacy and cosmetic industry. Employing cell cultures to produce these compounds industrially have been accomplished via a two stage culture system. The first stage, using a B5 cell-growth medium, was applied for cells growth without shikonin production. The second stage was then implemented using a M9 production medium to quickly induce the formation of shikonin and its derivatives. This special system provides a promising model for revealing the evoked mechanism of the biosynthesis of secondary metabolites such as shikonin and its derivatives. Further researches on this evoked mechanism will lay foundations for the bioengineering application on secondary metabolism in Boraginaceae plants.Shikonin and its derivatives are biosynthetically derived from two key precursors p-hydroxybenzoic acid (PHB), formed from phenylpropanoid metabolites, and geranyl pyrophosphate (GPP), derived from the isoprenoid pathway in cultured L. erythrorhizon cells. In B5 medium, the cultured cells accumulate in their vacuoles a large amount of p-hydroxybenzoic acid-O-glucoside, whose aglycone is one of the precursors of shikonin biosynthesis. On transfer of the cells from B5 to M9 medium, the glucoside is enzymatically hydrolyzed to give free hydroxybenzoic acid, which is then prenylated to form m-geranyl-p-hydroxybenzoic acid, a key intermediate leading to shikonin, as reviewed by Tabata in detail. Although the biochemistry and enzymology of the shikonin biosynthesis pathway are well understood, and many key enzymatic genes have been cloned and characterized, the molecular regulatory mechanism of shikonin biosynthesis was largely unknown.MYB、MYC transcription factors (TFs) regulate the activity of some branches of phenylpropanoid metabolism. The plant-specific R2R3 type MYB TFs family is defined by a common DNA-binding domain of two repeats of about 50 amino acids. MYC genes called basic helix-loop-helix (bHLH) encode a group of functionally diverse transcription factors found in plants and animals. MYB、MYC play important roles in the regulation of particular branches of phenylpropanoid metabolism, such as anthocyanin production, phlobaphene biosynthesis, flavonol biosynthesis, hydroxycinnamic acid biosynthesis, and monolignol biosynthesis. In view of the common phenylpropanoid metabolism pathway of shikonin biosynthesis, we think that these transcription factors may play an important role in regulating shikonin biosynthesis.A PCR-based method was used to isolate R2R3 type MYB genes from cultured cells of L. erythrorhizon. We have isolated and cloned three full-length cDNA sequences of R2R3 type MYB genes, named as LeMYBl, LeMYB2 and LeMYB3, and encoded 212,318 and 270 amino acids with an estimated molecular mass of 24.55 kDa,35.61 kDa and 30.40 kDa and a calculated p1 of 9.32, 8.64 and 8.56, respectively. A phylogenetic analysis of the R2R3 region of this conserved deduced amino acid sequence showed that LeMYBl belonged to subfamily 2 motif of the A. thaliana R2R3 type MYB TF classification which was involved in jasmonic acid and regulated phenylpropanoid biosynthetic pathway or indolic glucosinolates. Phylogenetic trees constructed with sequences from Arabidopsis thaliana AtMYB4 and strawberry FaMYB1 group the LeMYB2, LeMYB3 sequences with the subfamily 4, which was characterized with transcript repressor motif. It is proposed that in L. erythrorhizon cell commitment to synthesize shikonin relies on the balance between the activity of activator and repressor MYBs.LeMYC is also a novel member of the bHLH family that was cloned from Lithospermum erythrorhizon by using rapid amplification of cDNA ends method. Bioinformatics analyses showed that the predicted LeMYC protein contained a potential bHLH domain, was highly homologous to AtMYC2 from Arabidopsis thaliana, and encoded 636 amino acids with an estimated molecular mass of 69.8 kDa and a calculated p1 of 5.78. Phylogenetic analysis indicated LeMYC belong to subfamily III (d+e) of A. thaliana bHLH TF classification, which is involved in abscisic acid, jasmonic acid (JA), light signaling and phenylpropanoid biosynthetic pathways.In addition, we isolated and cloned the promoter sequences of MYB、MYC genes. The promoter sequences were analyzed for putative cis-acting elements using the PLACE database. This analysis revealed a number of potential regulatory motifs corresponding to several known cis-acting elements related to tissue-specific gene expression, abiotic- and biotic-stress responses were predicted. Especially, some important cis-acting elements related to shikonin formation were involved in the promoter region, for example, Methyl jasmonate response elements and Cu2+ response elements were also found, which suggested that MYB、MYC gene was involved in MeJA and Cu2+ signal transduction.We analyzed the expression patterns of MYB、MYC genes by using real-time PCR method. MYB、MYC was significantly induced transiently during the early stage when L. erythrorhizon cells were transferred from a B5 to an M9 medium to form shikonin. Exogenous methyl jasmonate (MeJA), an effective inducer of shikonin accumulation, also remarkably induced the rapid MYB、MYC expression. By contrast, IBU, an inhibitor of jasmonate biosynthesis, significantly inhibited MYB、MYC expression.2,4-D, an inhibitor of shikonin formation, could down-regulated the expression of LeMYB genes, but did not influence LeMYC transcript. Tissue-specific expression analysis showed that MYB、MYC mRNA predominantly accumulated in the roots where shikonin was biosynthesized. These results indicated that MYB、MYC gene could be novel members of the MYB、MYC gene family and may have an important function in shikonin formation.For primarily exploring the function of the LeMYB1, we transform the LeMYBl into Arabidopsis thaliana and gain transgenic lines. Phenotypic analysis showed that in T1 generation of transgenic Arabidopsis thaliana the leaves color turn purple, stem color become dark, and the whole transgenic line showed marked enhancement of anthocyanins.A simple and rapid protocol for induction of Agrobacterium rhizogenes-mediated hairy root in Lithospermum erythrorhizon was developed through infection of seedling internodes with needle-sticking method. The axenic hairy root, which confirmed by amplying the rolC gene by PCR, were proliferated under darkness in hormone-free solid B5 medium and RC liquid medium, showing multiple branching and plagiotropism. When hairy roots were subcultured to M9 or phytohormone free RC liquid medium, a large amount of shikonin and its derivatives were produced and released to the medium. This is the first report for inducing hairy root in L. erythrorhizon by using a kind of "infection of seedling internodes with needle-sticking method". This system provides foundation for producing large amount of shikonin industrially. Based on this established transgenic system of L. erythrorhizon, we obtained transgenic LeMYB1 hairy roots. Primary results of shikonin production showed pigments content of the transgenic LeMYB1 hairy roots were higher than wild type. This result indicated that LeMYB1 plays an important role in promoting shikonin biosynthesis. The subsequent researches will reveal the molecular mechanism of LeMYBl in promoting shikonin biosynthesis. The studies obtained hairy roots of L. erythrorhizon by stabbing into the seedling internodes with a sharp needle, which lays foundations for large-scale production of shikonin.In conclusion, this study not only facilitates our understanding about the molecular regulatory mechanism of secondary metabolism, but also lays foundations for the bioengineering application on efficiently regulating secondary metabolism in plants. |
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