Regulation of constitutive and inducible metabolites in 'hairy root' cultures of Hyoscyamus muticus

"Hairy root" cultures of Hyoscyamus muticus have been used to study the regulation of constitutive and inducible metabolites. The model systems were the alkaloid hyoscyamine and the sesquiterpenes lubimin and solavetivone.; In an effort to increase hyoscyamine production a system based on the selection for overproduced metabolic precursors (putrescine and phenylalanine) was established. Hairy roots were converted into cell suspensions and screened for resistance to p-fluorophenylalanine (PFP). Hairy roots were regenerated from both PFP-sensitive and PFP-resistant cells. Almost 2-fold increase in hyoscyamine levels were found in hairy roots regenerated from PFP-resistant cells.; Biosynthetically, sesquiterpenes are derived from acetate. Similarly, acetate is incorporated into the nitrogen ring of hyoscyamine. {dollar}sp{lcub}14{rcub}{dollar}C-acetate was added to hairy root cultures of H. muticus and the changes in incorporation of this common precursor into hyoscyamine and sesquiterpenes (lubimin and solavetivone) under fungal elicited and non-elicited conditions was determined. Almost 25% of the fed {dollar}sp{lcub}14{rcub}{dollar}C-acetate was incorporated into lubimin upon elicitation. Incorporation into hyoscyamine was much lower and not significantly different in elicited vs. non-elicited conditions. Hyoscyamine yields remained almost the same under elicited vs. non-elicited conditions.; To better understand the regulation of sesquiterpene biosynthesis a 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) cDNA from Cathoranthus roseus was introduced into Hyoscyamus muticus via Agrobacterium rhizogenes-mediated transformation. Two transgenic hairy root lines were obtained (HMGR-1 and HMGR-2). Under non-elicited conditions the levels of HMGR for the control remained almost stable at 1200 pmol/h/mg protein. In the transgenic lines the levels were also stable, being higher in the HMGR-2 line (between 4000 and 5000 pmol/h/mg). Interestingly, HMGR-1 showed lower HMGR levels than the control. Upon addition of elicitor, the levels of HMGR were induced by almost 3-fold in the control. None of the transgenic lines showed HMGR activation upon elicitation. Overall, total sesquiterpenes were higher in the HMGR-2 line which also showed an increased HMGR activity. However, these levels were not significantly different from those of previously established hairy root lines, suggesting that other factors such as channelling may be also important in regulating the biosynthesis of these sesquiterpenes.