In vivo nuclear-magnetic-resonance analysis of polyamine and alkaloid metabolism in transformed root cultures of Datura stramonium L.:: evidence for the involvement of putrescine in phytohormone-induced de-differentiation

The utilisation and accumulation of (15)N-labeled metabolites by a (15)N-labeled transformed root culture of Datura a stramonium L. was investigated by in vivo (15)N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [(15)N]glutamate and [(15)N]glutamine were rapidly depleted and there was an increase in the (15)N-NMR signals from conjugated putrescines and hyoscyamine. The signal from the conjugated putrescines passed through a maximum 2 d after the roots were resuspended, and it was concluded that putrescine could be stored as putrescine conjugates prior to its utilisation in other pathways. The transient accumulation of (15)N-label in the hydroxy-cinnamoylputrescines was reduced when the de-differentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing alpha-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-differentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, DL-alpha-difluoromethylarginine and DL-alpha-difluoromethylornithine. In-vivo (15)N-NMR spectra of roots grown in (15)N-enriched medium supplemented with these inhibitors showed that the (15)N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. DL-alpha-difluoromethylarginine also prevented the phytohormone induced de-differentiation of the root cultures, and this effect could be reversed by the supply of exogenous putrescine. Thus the supply of putrescine appears to play a crucial role in mediating the phytohormone induced de-differentiation of the root culture.