Hyoscyamine and proline accumulation in water-stressed hyoscyamus muticus 'hairy root' cultures

The patterns of hyoscyamine and proline accumulation were studied in Agrobocterium-transformed 'hairy root' cultures of Hyoscyamus muticus to determine if proline is a metabolic precursor of hyoscyamaine. Root cultures were stressed osmotically with mannitol and the subsequent growth, hyoscyamine levels, and proline levels were measured after ca, each transfer to fresh experimental medium for a total of four transfers. H. muticus 'hairy roots' were also treated with [U-14C proline or [1,4-14C] putrescine and analyzed for radioactive hyoscyamine. Growth of 'hairy root' cultures was reduced up to 90% in 0.4 M mannitol, and this inhibition persisted for at least four transfers. 'Hairy root' cultures of H. muticus accumulated hyoscyamine and free proline (up to 6-fold and 25-fold, respectively) when osmotically stressed with mannitol and this effect also persisted for four transfers when grown in the same mannitol concentration. Because the total production of hyoscyamine was also increased by twofold, we conclude that the elevated hyoscyamine concentration results from increased hyoscyamine synthesis and not from reduced growth. H. muticus 'hairy roots' incorporated radioactivity from [1,414C] putrescine efficiently into hyoscyamine in both treatments, but failed to convert [U-14C] proline into hyoscyamine. We thus conclude that accumulated proline does not serve as a precursor for hyoscyamine.