A number of genes that function in the terpenoid indole alkaloids (TIAs) biosynthesis pathway have been identified in Catharanthus roseus . Except for the geraniol 10-hydroxylase (G10H) gene, which encodes a cytochrome P450 monooxygenase, several of these genes are up-regulated by ORCA3, a jasmonate-responsive APETALA2 (AP2)-domain transcript factor. In this study, the G10H gene was transformed independently, or co-transformed with ORCA3 into C. roseus , using Agrobacterium rhizogenes MSU440. Hairy root clones expressing the G10H gene alone, or both the G10H and ORCA3 genes, were obtained. Alkaloid accumulation level analyses showed that all transgenic clones accumulated more catharanthine, with the highest accumulation level in the transgenic clone OG12 (6.5-fold higher than that of the non-expression clone). Following treatment with ABA, accumulation of catharanthine reached 1.96 mg/g DW in the transgenic clone OG12. The expression levels of TIAs biosynthesis genes in transgenic and non-transgenic clones were also investigated.
Vallesiachotamine and isovallesiachotamine are pharmacologically active monoterpene indole alkaloids produced by Catharanthus roseus. However, the biosynthetic pathway for these two alkaloids has not been characterized. The results of our experiments demonstrated that catharanthine induced the biosynthesis of vallesiachotamine and isovallesiachotamine in C. roseus cambial meristematic cells (CMCs) in a time- and dosage-dependent manner. The highest yields of vallesiachotamine and isovallesiachotamine (i.e., 1.49 mg l−1 and 1.08 mg l−1, respectively) were observed in CMCs treated with 10 mg catharanthine l−1 for 12 h. The cells growth was inhibited by 20 mg catharanthine l−1 (i.e., 16.3% decrease in dry-cell weight). A quantitative reverse transcription polymerase chain reaction analysis revealed that catharanthine regulated the biosynthesis of vallesiachotamine and isovallesiachotamine by up-regulating the expression of specific genes, including STR (strictosidine synthase), SGD (strictosidine β-D-glucosidase) and ORCA3 (transcription factor octadecanoid-responsive Catharanthus AP2/ERF-domain 3). Finally, considering the up-regulated expression of STR and SGD was consistent with the accumulation of vallesiachotamine and isovallesiachotamine, a potential vallesiachotamine and isovallesiachotamine biosynthetic pathway was proposed.
Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus , and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.
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