Gene expression in tobacco low-nicotine mutants.

Abstract

Two nuclear genes, Nicl and Nic2, regulate nicotine levels in tobacco. nicl and nic2 are semidominant mutations in Burley 21 that reduce leaf nicotine levels and the activities of multiple enzymes in the nicotine pathway and simultaneously increase polyamine levels in cultured roots. Cultured roots homozygous for both mutations were used to isolate two cDNAs by subtraction hybridization; the transcript levels of these two cDNAs were much lower in the mutant roots than in the wild-type roots. The A411 gene encodes a 41-kD protein with considerable homology to mammalian spermidine synthase, whereas the A622 gene encodes a 35-kD protein with high homology to isoflavone reductase. When these genes were expressed in Escherichia coli, A411 had no spermidine synthase activity but did show putrescine N-methyltransferase activity, which is the first enzyme committed to the nicotine biosynthetic pathway, and A622 did not show isoflavone reductase activity. Both the methyltransferase and A622 genes are predominantly expressed in the root, and their expression levels in cultured roots are coordinately decreased by the nic mutations in the order of wild type > nic2 > nicl > nicl nic2. Removal of tobacco flower heads and young leaves rapidly and coordinately induced both genes in the root. Further, exogenous supply of auxin down-regulated both genes in cultured tobacco roots. These results suggest that N i c l and Nic2 are regulatory genes for nicotine biosynthesis.

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

“…S2 illustrates that S. pombe cells with an empty vector, and those cells with a subcloned NUP1 cDNA accumulated comparable 3 H-adenine levels. In contrast, yeast cells harboring the NUP1 cDNA showed significantly more 14 C-nicotine uptake activity (Fig. 1A) relative to vector controls.…”

“…Restricted root growth in pot-bound plants eliminates an inducible nicotine chemical defense (5). Regulation of nicotine biosynthesis (12,13) and nicotine biosynthetic gene expression (8,(14)(15)(16)(17)(18) are under the coordinate regulation of the A and B loci, also called NIC1 and NIC2, respectively (14). In addition to regulating nicotine biosynthetic genes, the A and B loci regulate a large and complex regulon of apparent stress response genes that are unrelated to alkaloid biosynthesis (15).…”

Tobacco Nicotine Uptake Permease Regulates the Expression of a Key Transcription Factor Gene in the Nicotine Biosynthesis Pathway

“…Quinolinate phosphoribosyltransferase (QPT) catalyzes the last step of this pathway to yield nicotinate mononucleotide (Katoh et al, 2006), and one of the duplicated QPT genes functions in nicotine formation, rather than in NAD biosynthesis (Shoji and Hashimoto, 2011b). The pyrrolidine ring of nicotine is synthesized from Orn by three enzymes: Orn decarboxylase, putrescine N-methyltransferase (PMT; Hibi et al, 1994), and N-methylputrescine oxidase.…”

Nicotine biosynthesis is regulated by two more layers: Small and long non-protein-coding RNAs

“…12,31,32 Genes involved in rate-limiting steps or at metabolic branch points are the most common targets for genetic modifications. 23,29,31,33 To reduce the content of certain alkaloid by interrupting the rate-limiting gene is relatively easy. 23,33 However, increasing levels of a certain alkaloid by overexpressing one or several key genes remains a real challenge 2 because of emerging new rate-limiting steps in the metabolic pathways and some unknown factors.…”

“…23,29,31,33 To reduce the content of certain alkaloid by interrupting the rate-limiting gene is relatively easy. 23,33 However, increasing levels of a certain alkaloid by overexpressing one or several key genes remains a real challenge 2 because of emerging new rate-limiting steps in the metabolic pathways and some unknown factors. Fully understanding how alkaloid metabolic pathways are regulated, and exploring other ways to manipulate them are essential for improving alkaloid production by metabolic engineering.…”