Antisense-mediated reduction in ADC activity causes minor alterations in the alkaloid profile of cultured hairy roots and regenerated transgenic plants of Nicotiana tabacum

Abstract

In species of the genus Nicotiana, as in most plants, the important polyamine precursor putrescine can be derived from the amino acids ornithine and/or arginine via the activity of ornithine decarboxylase (ODC) and/or arginine decarboxylase (ADC), respectively. Nicotiana species also utilize putrescine to provide the pyrollidine ring of the defensive alkaloid nicotine and its derivatives. Previous biochemical studies, involving callus tissues cultured in vitro, suggested that the ADC-mediated route to putrescine is used preferentially to provide the putrescine that is utilized for nicotine synthesis in N. tabacum. To ascertain if this is the case in N. tabacum plants, where nicotine synthesis takes place exclusively in roots, we used an antisense approach to diminish ADC activity in transformed roots which were cultured in vitro. Several independent lines were recovered possessing markedly reduced levels of ADC transcript and ADC activity compared to controls. Transcript levels of other genes in this general area of metabolism, including ODC, were not altered as a result of the antisense-mediated downregulation of ADC. Concentrations of nicotine were comparable in antisense-ADC and control hairy root lines throughout most of their respective culture cycles, except at the latter stages of growth when the nicotine content of antisense-ADC hairy root lines was observed to be approximately 20% lower than in controls. Levels of anatabine, the second most abundant alkaloid typically found in N. tabacum, which is not derived from putrescine, were slightly elevated in two antisense-ADC hairy root lines at the latter stages of their culture cycles compared to controls. Comparison of alkaloid levels in leaves of transgenic plants that were regenerated from separate antisense-ADC and control transformed root lines indicated that the former possessed slightly elevated levels of anatabine but did not contain average levels of leaf nicotine that were different from that of controls. Our overall conclusion is that the ADC mediated route to putrescine plays a role, but is not of prime importance, in providing the pyrollidine ring which is used for nicotine synthesis in cultured hairy roots of N. tabacum and in roots of healthy greenhouse-grown plants.