Molecular analysis of alkaloid metabolism in AABB v. aabb genotype Nicotiana tabacum in response to wounding of aerial tissues and methyl jasmonate treatment of cultured roots

Abstract

Synthesis of the wound-inducible alkaloid, nicotine, in roots of the allotetraploid species Nicotiana tabacum L. is strongly influenced by the presence of two non-allelic genes, A and B. Together, these loci affect baseline transcript levels of genes dedicated to secondary metabolism (e.g. PMT and A622) as well as genes with roles in separate areas of primary metabolism (e.g. ODC, ADC, SAMS — polyamines; QPT — pyridine nucleotide cycle). Experiments comparing high alkaloid variety NC 95 (AABB genotype) and near-isogenic low alkaloid N. tabacum variety LAFC 53 (aabb genotype) indicate that together, mutations in the A and B loci diminish, but do not ablate, the propensity of roots to increase transcript levels of genes involved in alkaloid metabolism after damage to aerial tissues or direct treatment with the wound hormone, methyl jasmonate. Accordingly, roots of aabb genotype can increase their nicotine content somewhat in response to these treatments. Additionally, we show that transcript levels of genes associated with polyamine metabolism (ODC, ADC, SamDC, SAMS and SS) but not alkaloid synthesis (PMT, QPT, A622) are elevated in leaves of N. tabacum in response to wounding. Moreover, respective increases in transcript levels of each gene are similar in wounded leaves of NC 95 and LAFC 53, suggesting that these increases are not controlled by combined action of genes encoded by the A and B loci. Further detailed analysis of wounded leaves of AABB genotype indicates that although transcript levels of these genes of polyamine metabolism and associated enzyme activities for ODC, ADC and SamDC, are markedly increased in leaves in response to wounding, putrescine levels remain unaltered whilst spermidine and spermine levels are reduced to 50–60% of controls levels, when analysed up to 6 h post-wounding. These observations may indicate that any wound-induced increases in polyamine biosynthesis that do occur in leaf cells during this time frame are consumed by metabolic reactions involved in repair and / or strengthening of wounded leaf tissues.

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

“…First, we analysed ORC1 expression in whole organs of hydroponically grown plants. Consistent with nicotine biosynthesis taking place in the roots of tobacco plants, the key biosynthetic genes PMT and QPRT are preferentially transcribed in root tissues (Shoji et al, 2000;Sinclair et al, 2000;Cane et al, 2005). In mock conditions, ORC1 transcript levels were lower in the leaves than in the roots (Figure 2).…”

“…The expression of structural genes involved in nicotine biosynthesis is stimulated by methyl jasmonate (MeJA) in cultured cells or hairy roots of Nicotiana species (Imanishi et al, 1998;Goossens et al, 2003;Cane et al, 2005). These genes include those coding for ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and putrescine N-methyltransferase (PMT), all three essential for the synthesis of the pyrroline ring, as well as aspartate oxidase (AO) and quinolinate phosphoribosyltransferase (QPRT), essential for the synthesis of the pyridine ring.…”

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

“…7). Previous studies show a good correlation of concerted increases in the expression of multiple nicotine biosynthetic genes with increases in nicotine accumulation levels (8,39,40), indicating metabolic control mediated by the abundance of transcripts encoding nicotine biosynthetic enzymes. In contrast, the NUP1-reduced expression lines did not show a good correlation of five nicotine biosynthetic gene expression levels with reduced nicotine accumulation levels, suggesting that NUP1 may influence mechanisms of alkaloid metabolic control other than transcript abundance levels.…”

“…Foliar wounding stimulates the production of jasmonic acid derivatives (7), such as methyljasmonic acid (MeJA), and these derivatives are transported to the roots through the phloem (6). Exogenous MeJA treatment alone is sufficient to increase nicotine biosynthetic levels (7) and increase the mRNA accumulation levels of transcripts encoding nicotine biosynthetic enzymes (8).…”

“…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).…”