SUMMARYTranscription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

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.

SummaryCyanogenic glucosides are present in several crop plants and can pose a significant problem for human and animal consumption, because of their ability to release toxic hydrogen cyanide. Sorghum bicolor L. contains the cyanogenic glucoside dhurrin. A qualitative biochemical screen of the M2 population derived from EMS treatment of sorghum seeds, followed by the reverse genetic technique of Targeted Induced Local Lesions in Genomes (TILLING), was employed to identify mutants with altered hydrogen cyanide potential (HCNp). Characterization of these plants identified mutations affecting the function or expression of dhurrin biosynthesis enzymes, and the ability of plants to catabolise dhurrin. The main focus in this study is on acyanogenic or low cyanide releasing lines that contain mutations in CYP79A1, the cytochrome P450 enzyme catalysing the first committed step in dhurrin synthesis. Molecular modelling supports the measured effects on CYP79A1 activity in the mutant lines. Plants harbouring a P414L mutation in CYP79A1 are acyanogenic when homozygous for this mutation and are phenotypically normal, except for slightly slower growth at early seedling stage. Detailed biochemical analyses demonstrate that the enzyme is present in wildtype amounts but is catalytically inactive. Additional mutants capable of producing dhurrin at normal levels in young seedlings but with negligible leaf dhurrin levels in mature plants were also identified. No mutations were detected in the coding sequence of dhurrin biosynthetic genes in this second group of mutants, which are as tall or taller, and leafier than nonmutated lines. These sorghum mutants with reduced or negligible dhurrin content may be ideally suited for forage production.

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.

Nicotiana tabacum L. produces a number of pyridine alkaloids, with nicotine representing the major component and anatabine comprising most of the remainder of the alkaloid fraction. An antisense approach was used here to down-regulate activity of the important enzyme putrescine N-methyltransferase (PMT) in transformed roots of this species to determine effects upon alkaloid metabolism. Transformed root lines were produced that contained markedly reduced PMT activity, with a concomitant reduction in nicotine content compared to controls. No negative effects upon growth were observed. Several antisense-PMT transformed root lines, and also leaf tissues of regenerated transformed plants, showed a substantial increase in anatabine content relative to controls. Northern hybridization experiments indicated that the antisense-PMT manipulation had little or no effect upon the transcript levels of other genes encoding enzymes involved in alkaloid metabolism, including quinolinate acid phosphoribosyltransferase (QPT). The latter enzyme plays a key role in regulating the synthesis of nicotinic acid which supplies the pyridine ring necessary for both nicotine and anatabine synthesis. We suggest that elevated anatabine levels in antisense-PMT lines are a direct consequence of a relative oversupply of nicotinic acid which, in the absence of adequate levels of 1-methyl-delta(1)-pyrrolinium cation (the ultimate product of PMT activity), is used to synthesise anatabine directly. As is discussed, no naturally occurring species or varieties of Nicotiana are known that typically produce high levels of anatabine in root or leaf tissues, meaning that the antisense PMT transgenics produced in this study have no natural counterpart. These experiments thus represent an example of metabolic engineering of plant pyridine metabolism, via antisense down-regulation of gene expression in a contributing pathway leading to secondary metabolite biosynthesis.