The A622 gene in Nicotiana glauca (tree tobacco): evidence for a functional role in pyridine alkaloid synthesis

Abstract

Nicotiana glauca (Argentinean tree tobacco) is atypical within the genus Nicotiana, accumulating predominantly anabasine rather than nicotine and/or nornicotine as the main component of its leaf pyridine alkaloid fraction. The current study examines the role of the A622 gene from N. glauca (NgA622) in alkaloid production and utilises an RNAi approach to down-regulate gene expression and diminish levels of A622 protein in transgenic tissues. Results indicate that RNAi-mediated reduction in A622 transcript levels markedly reduces the capacity of N. glauca to produce anabasine resulting in plants with scarcely any pyridine alkaloids in leaf tissues, even after damage to apical tissues. In addition, analysis of hairy roots containing the NgA622-RNAi construct shows a substantial reduction in both anabasine and nicotine levels within these tissues, even if stimulated with methyl jasmonate, indicating a role for the A622 enzyme in the synthesis of both alkaloids in roots of N. glauca. Feeding of Nicotinic Acid (NA) to hairy roots of N. glauca containing the NgA622-RNAi construct did not restore capacity for synthesis of anabasine or nicotine. Moreover, treatment of these hairy root lines with NA did not lead to an increase in anatabine levels, unlike controls. Together, these results strongly suggest that A622 is an integral component of the final enzyme complex responsible for biosynthesis of all three pyridine alkaloids in Nicotiana.

Clustered Transcription Factor Genes Regulate Nicotine Biosynthesis in Tobacco

“…Nicotine is composed of a pyridine ring and a pyrrolidine ring (see Supplemental Figure 1 online); ornithine decarboxylase (ODC; Imanishi et al, 1998), putrescine N-methyltransferase (PMT; Hibi et al, 1994), and N-methylputrescine oxidase (MPO; Heim et al, 2007;Katoh et al, 2007) are involved in the formation of the pyrrolidine ring, whereas the pyridine ring uses enzymes involved in the early steps of NAD biosynthesis, such as aspartate oxidase (AO), quinolinic acid synthase (QS), and quinolinic acid phosphoribosyl transferase (QPT) (Sinclair et al, 2000;Katoh et al, 2006). A PIP family oxidoreductase, A622, is also required for the biosynthesis of tobacco alkaloids, possibly to form a nicotinic acid-derived precursor (Deboer et al, 2009;Kajikawa et al, 2009). Biosynthesis of nicotine and nornicotine requires both pyrrolidine and pyridine pathways, whereas anatabine and anabasine are synthesized from the pyridine pathway alone .…”

DNA-binding and transcriptional activation properties of tobacco <i>NIC2</i>-locus ERF189 and related transcription factors

“…Insect herbivory on tobacco leaves triggers a jasmonatemediated signaling cascade, which results in increased formation of nicotine in the roots and subsequent transport of nicotine to the aerial parts (Shoji et al 2008;Shoji and Hashimoto 2011c). Jasmonate-or herbivoryinduced tobacco genes include structural genes encoding enzymes and transporters involved in nicotine biosynthesis and accumulation, such as ornithine decarboxylase (ODC; Imanishi et al 1998), putrescine N-methyltransferase (PMT; Hibi et al 1994), qunolinate phosphoribosyltransferase (QPT; Sinclair et al 2000), a PIP-family oxidoreductase A622 (De Boer et al 2009;Hibi et al 1994;Kajikawa et al 2009) and tonoplastlocalized multidrug and toxic compound extrusion (MATE)-type transporters MATE1 and MATE2 (Shoji et al 2009) (Figure 1). ese tobacco genes are expressed in restricted cell types in the roots, and are coordinately elicited in space and time by jasmonate (Shoji and Hashimoto 2011a).…”

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

“…As much as 60 mM of nicotine accumulates in the vacuoles of the leaf epidermal cells at the tip (Lochmann et al, 2001). Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the nicotine-specific pathway, and a PIP-family reductase, called A622, was also suggested to function in a late step in nicotine biosynthesis (Hibi et al, 1994;Shoji et al, 2000aShoji et al, , 2000bDeBoer et al, 2009;Kajikawa et al, 2009). PMT and A622 proteins are specifically expressed in the same cell types in the root (Shoji et al, 2000a(Shoji et al, , 2002.…”