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 of15N-labeled metabolites by a15N-labeled transformed root culture of Datura stramonium L. was investigated by in vivo15N-nuclear-magnetic-resonance (NMR) spectroscopy. After resuspension in spent growth medium, the pools of [15N]glutamate and [15N]glutamine were rapidly depleted and there was an increase in the15N-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 of15N-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 α-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-α-difluoromethylarginine and DL-α-difluoromethylornithine. In-vivo15N-NMR spectra of roots grown in15N-enriched medium supplemented with these inhibitors showed that the15N-labelling of the conjugated polyamines and hyoscyamine was markedly reduced. DL-α-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.