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

RNAi-mediated down-regulation of ornithine decarboxylase (ODC) leads to reduced nicotine and increased anatabine levels in transgenic Nicotiana tabacum L.

“…Previous work in this laboratory used an antisense approach to down-regulate ADC transcript levels in both hairy roots cultured in vitro, and in regenerated transgenic plants grown in soil. Despite a substantial decrease in both ADC transcript levels and enzyme activity, only minor effects upon the capacity of N. tabacum to synthesize nicotine were observed (Chintapakorn and Hamill, 2007). Together with previous observations that showed ODC transcript levels and enzymatic activity were strongly up-regulated in plants in response to wounding, and also in cell and root cultures treated with methyl jasmonate (Mizusaki et al, 1973;Saunders and Bush, 1979;Imanishi et al, 1998;Wang et al, 2000;Xu et al, 2004;Cane et al, 2005), these results suggested that the presence of ODC in N. tabacum is essential for nicotine production, particularly in response to wound-associated stress.…”

“…This was deemed to be important as preliminary experiments involving N. tabacum ODC sequences from the 5 0 region of the cDNA led to the detection of the primary, non-processed RNAi-construct in RNA from transgenic lines on northern filters (data not shown). ADC: 1.2 kb XbaI fragment representing part of the N. tabacum ADC cDNA (Accession number AF127241; details noted in Chintapakorn and Hamill, 2007). Ubiquitin: 1.8 kb EcoRI cDNA fragment representing the ubiquitin gene from Antirrhinum majus (courtesy of Prof. C. Martin, Norwich UK; details noted in Chintapakorn and Hamill, 2003).…”

“…Pyridine alkaloids were identified on the basis of their diode array spectrum and quantified at 260 nm using known amounts of authentic standards (Sigma). ODC and ADC enzyme assays were performed as outlined previously (Hamill et al, 1990;Cane et al, 2005;Chintapakorn and Hamill, 2007).…”

“…For many years, there was some debate in the scientific literature as to whether the ODC or the ADC route to putrescine is the more important in enabling nicotine production in Nicotiana (Tiburcio and Galston, 1986;Walden et al, 1997; also see Chintapakorn and Hamill, 2007 and references therein for a fuller discussion). Previous work in this laboratory used an antisense approach to down-regulate ADC transcript levels in both hairy roots cultured in vitro, and in regenerated transgenic plants grown in soil.…”

Polyamines delay leaf maturation in low‐alkaloid tobacco varieties

“…Previous studies have demonstrated that the ADC route to putrescine has only a minor effect on the alkaloid profile of tobacco whereas the ODC pathway plays the major role in nicotine biosynthesis (Chintapakorn & Hamill, 2007;Dalton et al, 2016;DeBoer, Dalton, Edward, & Hamill, 2011;DeBoer, Dalton, Edward, Ryan, & Hamill, 2013). Putrescine is converted to spermidine and then spermine by the successive addition of aminopropyl groups derived from decarboxylated S-adenosylmethionine (SAM), in reactions catalyzed by the enzymes spermidine synthase and spermine synthase, respectively.…”