Antisense-mediated down-regulation of putrescine N-methyltransferase activity in transgenic Nicotiana tabacum L. can lead to elevated levels of anatabine at the expense of nicotine

Abstract

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 contained 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-delta(1)-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.

Metabolic Engineering of Tropane Alkaloid Biosynthesis in Plants

“…Once the specific element involved in JERE gene expression is obtained, we can isolate the transcription factor using a yeast one-hybrid screening system with this element as bait, which will be of considerable importance and interest for the metabolic engineering of tropane. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites (Chintapakorn and Hamill 2003).…”

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

“…Transgenic regenerants were rooted in MS medium containing 25 mg L )1 kanamycin sulphate before being transferred to an insect-free greenhouse and selfed to produce T 1 seeds. Transformed N. glauca root cultures were created with the methodology outlined previously (Chintapakorn and Hamill, 2003;Cane et al, 2005). Clonal root cultures were maintained in the dark at 22°C, shaking at 100 r.p.m.…”

“…Transgene overexpression in transformed tobacco plants and tree tobacco root lines was verified by northern blot analysis as described (Chintapakorn and Hamill, 2003). DNA fragments used for molecular probes for TFs were recovered by RT-PCR on tobacco root RNA (see Table S1 for the primers).…”

“…DNA fragments used for molecular probes for TFs were recovered by RT-PCR on tobacco root RNA (see Table S1 for the primers). Molecular probe generation for Nicotiana rustica QPRT (accession number AJ243436) and Antirrhinum majus ubiquitin is described in Chintapakorn and Hamill (2003).…”

Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

“…In the alkaloid biosynthetic pathway, PMT catalyses the first committed step driving the flux of nitrogen from polyamine biosynthesis to alkaloid biosynthesis (Facchini, 2001, Robins et al, 1994. It is the rate-limiting enzyme in nicotine synthesis and its transcripts are found only in the roots (Chintapakorn and Hamill, 2003).…”