betadex and coronatine

Hyoscyamine (HYO) and scopolamine (SCO) are tropane alkaloids acting as anticholinergic factors on the parasympathetic nervous system in humans and are produced by Solanaceous plants. Two strains of Agrobacterium rhizogenes, A4 and LBA9402, were used to infect Atropa acuminata Royle ex Miers and Atropa belladonna L. leaf explants. A. acuminata was inoculated either by direct infection or sonicated-assisted A. rhizogenes-mediated transformation (SAAT) was performed. A. belladonna was inoculated with the A4 strain using a direct method. The selected hairy root lines of both species were elicited with 50 mM methyl-β-cyclodextrin (β-CD), 0.5 μM coronatine (Cor) or 50 mM β-CD + 0.5 μM Cor on Day 14 of culture. The elicitor effect on growth and HYO and SCO content was analyzed after one (T1) and two (T2) weeks of treatment. In A. acuminata explants, the highest transformation percentage (T%) was obtained with strain A4 and the SAAT method (T%: 96.43). Cor significantly reduced the growth of A. acuminata hairy roots (fresh weight and dry weight [DW]: 2.52 and 0.3 g, respectively), whereas β-CD increased their DW (0.4 g). Also, the combined β-CD + Cor treatment had a positive significant effect on the DW of A. belladonna hairy roots (0.41 g). In A. acuminata hairy roots, the HYO level was lower under Cor treatment than in the control at both sampling times. In contrast, the SCO content was increased 10-fold by Cor elicitation at T1 compared to the control (10.95 mg g

Topics: Agrobacterium; Amino Acids; Atropa; Atropa belladonna; beta-Cyclodextrins; Indenes; Plant Roots; Tropanes

Paclitaxel is a potent antitumor alkaloid widely used for the treatment of several cancer types. This valuable secondary metabolite naturally exists in the inner bark of Taxus species in very low amounts. The small-scale production of paclitaxel in Taxus cell cultures requires utilization of several elicitors.. The main objective of this work was to identify key genes that encode rate-limiting enzymes in paclitaxel biosynthesis pathway by investigating the possible relationship between paclitaxel production and a set of 13 involved genes' relative expression in Taxus baccata L. cell suspension cultures affected by coronatine and methyl-β-cyclodextrin.. In the present research, the most important key genes were identified using gene expression profiling evaluation and paclitaxel production assessment in Taxus baccata L. cell cultures affected by mentioned elicitors.. Gene expression levels were variably increased using methyl-β-cyclodextrin, and in some cases, a synergistic effect on transcript accumulation was observed when culture medium was supplemented with both elicitors. It was revealed that DBAT, BAPT, and DBTNBT are the most important rate-limiting enzymes in paclitaxel biosynthesis pathway in Taxus baccata L. cell suspension cultures under coronatine and methyl-β-cyclodextrin elicitation condition. Moreover, PAM was identified as one of the important key genes especially in the absence of β-phenylalanine. In cell cultures affected by these elicitors, paclitaxel was found largely in the culture media (more than 90%). The secretion of this secondary metabolite suggests a limited feedback inhibition and reduced paclitaxel toxicity for producer cells. It is the result of the ABC gene relative expression level increment under methyl-β-cyclodextrin elicitation and highly depends on methyl-β-cyclodextrin's special property (complex formation with hydrophobic compounds). Paclitaxel biosynthesis was obviously increased due to the effect of coronatine and methyl-β-cyclodextrin elicitation, leading to the production level of 5.62 times higher than that of the untreated cultures. Graphical abstract Rate Limiting Enzymes in Paclitaxel Biosynthesis Pathway: DBAT, BAPT, DBTNBT and PAM.

Topics: Amino Acids; beta-Cyclodextrins; Cell Culture Techniques; Cells, Cultured; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Indenes; Metabolic Networks and Pathways; Paclitaxel; Plant Proteins; Real-Time Polymerase Chain Reaction; Taxus