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Protocols in ecological and environmental plant physiology


Article << Previous     |     Next >>   Contents Vol 32(4)

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

Karen A. Cane A C, Melinda Mayer B, Angela J. Lidgett A, Anthony J. Michael B, John D. Hamill A D

A School of Biological Sciences, Monash University, Melbourne, Victoria 3800, Australia.
B Biotechnology and Biological Sciences Research Council, Institute of Food Research, Norwich Research Park, Norwich NR2 2AN, UK.
C Current address: Department of Primary Industries, PB 260, Horsham, Victoria 3401, Australia.
D Corresponding author. Email: john.hamill@sci.monash.edu.au
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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.

Keywords: mutant, nicotine, polyamine, tobacco, transcript, wound-induction.

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