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The utilisation and accumulation of 15 Nlabeled 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 Nlabel in the hydroxy-cinnamoylputrescines was reduced when the de-dierentiation of the root cultures into a suspension culture was initiated by exposure to a medium containing a-napthaleneacetic acid and kinetin. This led to the hypothesis that phytohormone-induced de-dierentiation of the root cultures required the presence of free polyamines, and this was tested using two potent inhibitors of putrescine biosynthesis, DL-a-di¯uor-omethylarginine and DL-a-di¯uoromethylornithine. Invivo 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-a-di¯uoro-methylarginine also prevented the phytohormone induced de-dierentiation of the root cultures, and this eect 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-dierentiation of the root culture.

The biosynthesis of the opine agropine in transformed Nicotiana tabacum L. root cultures was studied using in vivo N nuclear magnetic resonance (NMR) spectroscopy. Roots were incubated with [ N]ammonium and/or [ N]nitrate, and the incorporation of the label into agropine, conjugated polyamines, Γ‐aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln) and nicotine was monitored by NMR. The largest labelled pool was agropine in cells grown on a N‐enriched medium and its synthesis was maintained, to the detriment of the Gln pool, under conditions of nitrogen (N) starvation. These observations indicate that the synthesis of agropine is not tightly regulated and that it represents a significant sink for carbon (C) and N in the plant. The addition of α‐naphthaleneacetic acid (NAA) and kinetin to the growth medium caused de‐differentiation of the root cultures and perturbation of secondary N metabolism. The amount of agropine relative to Gln increased but the total accumulation of agropine decreased, in part because of the increase in the synthesis of conjugated polyamines. These results show that agropine biosynthesis perturbs both primary and secondary N metabolism, and that the perturbation differs according to the culture conditions and the imposed stress.