Plant Molecular Biology 53: 87–105, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Antisense-mediated down-regulation of putrescine N-methyltransferase
activity in transgenic Nicotiana tabacum L.canleadtoelevatedlevelsof
anatabine at the expense of nicotine
and John D. Hamill
School of Biological Sciences, Monash University, P.O. Box 18, Melbourne, Victoria 3800, Australia (*author
for correspondence; email email@example.com);
Current address: Department of Botany, Faculty of
Science, Chulalongkorn University, Bangkok 10330, Thailand
Received 16 May 2003; accepted in revised form 21 August 2003
Key words: anatabine, antisense, metabolism, nicotine, putrescine N-methyltransferase, pyridine alkaloid,
Nicotiana tabacum L. produces a number of pyridine alkaloids, with nicotine representing the major component
and anatabine comprising most of the remainder of the alkaloid fraction. An antisense approach was used here
to down-regulate activity of the important enzyme putrescine N-methyltransferase (PMT) in transformed roots
of this species to determine effects upon alkaloid metabolism. Transformed root lines were produced that con-
tained markedly reduced PMT activity, with a concomitant reduction in nicotine content compared to controls.
No negative effects upon growth were observed. Several antisense-PMT transformed root lines, and also leaf
tissues of regenerated transformed plants, showed a substantial increase in anatabine content relative to controls.
Northern hybridization experiments indicated that the antisense-PMT manipulation had little or no effect upon
the transcript levels of other genes encoding enzymes involved in alkaloid metabolism, including quinolinate acid
phosphoribosyltransferase (QPT). The latter enzyme plays a key role in regulating the synthesis of nicotinic acid
which supplies the pyridine ring necessary for both nicotine and anatabine synthesis. We suggest that elevated
anatabine levels in antisense-PMT lines are a direct consequence of a relative oversupply of nicotinic acid which,
in the absence of adequate levels of 1-methyl-
-pyrrolinium cation (the ultimate product of PMT activity), is used
to synthesise anatabine directly. As is discussed, no naturally occurring species or varieties of Nicotiana are known
that typically produce high levels of anatabine in root or leaf tissues, meaning that the antisense PMT transgenics
produced in this study have no natural counterpart. These experiments thus represent an example of metabolic
engineering of plant pyridine metabolism, via antisense down-regulation of gene expression in a contributing
pathway leading to secondary metabolite biosynthesis.
Abbreviations: ADC, arginine decarboxylase; GC/MS, gas chromatography/ mass spectrometry, GUS, β-
glucuronidase; HPLC, high-performance liquid chromatography; ODC, ornithine decarboxylase; PMT, pu-
trescine N-methyltransferase; QPT, quinolinate phosphoribosyltransferase; SAMDC, S-adenosylmethionine de-
carboxylase; SAMS, S-adenosylmethionine synthase; SPDS, spermidine synthase
The presence of pyridine alkaloids is a characteristic
feature of Nicotiana species (Dawson, 1962) and re-
cent studies suggest that they play an important role
in protecting plants against herbivory in their native
habitats (Baldwin, 1988, 1989; Voelckel et al., 2001).
Leaf tissues of most wild Nicotiana species contain
predominantly either nicotine or its derivative nor-
nicotine, with anabasine being a major constituent
of the alkaloid fraction in a limited number of spe-
cies (Saitoh et al., 1985; Sisson and Severson, 1990).