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
Ford YY., Ratcliffe RG., Robins RJ.
The utilisation and accumulation of 15 N-labeled metabolites by a 15 N-labeled transformed root culture of Datura 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 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-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-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.