Effects of down-regulating ornithine decarboxylase upon putrescine-associated metabolism and growth in Nicotiana tabacum L.

Transgenic plants of Nicotiana tabacum L. homozygous for an RNAi construct designed to silence ornithine decarboxylase (ODC) had significantly lower concentrations of nicotine and nornicotine, but significantly higher concentrations of anatabine, compared with vector-only controls. Silencing of ODC also led to significantly reduced concentrations of polyamines (putrescine, spermidine and spermine), tyramine and phenolamides (caffeoylputrescine and dicaffeoylspermidine) with concomitant increases in concentrations of amino acids ornithine, arginine, aspartate, glutamate and glutamine. Root transcript levels of S-adenosyl methionine decarboxylase, S-adenosyl methionine synthase and spermidine synthase (polyamine synthesis enzymes) were reduced compared with vector controls, whilst transcript levels of arginine decarboxylase (putrescine synthesis), putrescine methyltransferase (nicotine production) and multi-drug and toxic compound extrusion (alkaloid transport) proteins were elevated. In contrast, expression of two other key proteins required for alkaloid synthesis, quinolinic acid phosphoribosyltransferase (nicotinic acid production) and a PIP-family oxidoreductase (nicotinic acid condensation reactions), were diminished in roots of odc-RNAi plants relative to vector-only controls. Transcriptional and biochemical differences associated with polyamine and alkaloid metabolism were exacerbated in odc-RNAi plants in response to different forms of shoot damage. In general, apex removal had a greater effect than leaf wounding alone, with a combination of these injury treatments producing synergistic responses in some cases. Reduced expression of ODC appeared to have negative effects upon plant growth and vigour with some leaves of odc-RNAi lines being brittle and bleached compared with vector-only controls. Together, results of this study demonstrate that ornithine decarboxylase has important roles in facilitating both primary and secondary metabolism in Nicotiana.

RNAi-mediated down-regulation of ornithine decarboxylase (ODC) impedes wound-stress stimulation of anabasine synthesis in Nicotiana glauca.

Unlike most Nicotiana species, leaf tissues of the globally significant weed Nicotiana glauca Grah. (Argentinian tree tobacco) contains anabasine as the main component of its alkaloid pool, with concentrations typically increasing several fold in response to wounding of plants. The Δ(1)-piperidinium ring of anabasine is synthesised from cadaverine, via the decarboxylation of lysine, however the identity of the protein catalysing this reaction remains unknown. Recent studies indicate that ornithine decarboxylase (ODC), an enzyme involved in the synthesis of the diamine putrescine, may also possess LDC activity. Previously we found that ODC transcript is markedly up-regulated in leaves of N. glauca in response to wounding. In order to examine the role of ODC in the synthesis of anabasine in N. glauca, transcript levels were constitutively down-regulated in hairy root cultures and transgenic plants via the introduction of a CaMV35S driven ODC-RNAi construct. In addition to the anticipated marked reduction in nicotine concentrations, demonstrating that the ODC-RNAi construct was functioning in vivo, we observed that N. glauca ODC-RNAi hairy root cultures had a significantly diminished capacity to elevate anabasine synthesis in response to treatment with the wound-associated hormone methyl jasmonate, when compared to vector-only controls. We observed also that ODC-RNAi transgenic plants had significantly reduced ability to increase anabasine concentrations following removal of the plant apex. We conclude that ODC does have an important role in enabling N. glauca to elevate levels of anabasine in response to wound-associated stress.

RNAi-mediated down-regulation of ornithine decarboxylase (ODC) leads to reduced nicotine and increased anatabine levels in transgenic Nicotiana tabacum L.

In leaf and root tissues of Nicotiana tabacum L. (common tobacco), nicotine is by far the predominant pyridine alkaloid, with anatabine representing only a minor component of the total alkaloid fraction. The pyrrolidine ring of nicotine is derived from the diamine putrescine, which can be synthesized either directly from ornithine via the action of ODC, or from arginine via a three enzymatic step process, initiated by ADC. Previous studies in this laboratory have shown that antisense-mediated down-regulation of ADC transcript levels has only a minor effect upon the alkaloid profile of transgenic N. tabacum. In the present study, RNAi methodology was used to down-regulate ODC transcript levels in N. tabacum, using both the Agrobacterium rhizogenes-derived hairy root culture system, and also disarmed Agrobacterium tumefaciens to generate intact transgenic plants. We observed a marked effect upon the alkaloid profile of transgenic tissues, with ODC transcript down-regulation leading to reduced nicotine and increased anatabine levels in both cultured hairy roots and intact greenhouse-grown plants. Treatment of ODC-RNAi hairy roots with low levels of the wound-associated hormone methyl jasmonate, or wounding of transgenic plants by removal of apices - both treatments which normally stimulate nicotine synthesis in tobacco - did not restore capacity for normal nicotine synthesis in transgenic tissue but did lead to markedly increased levels of anatabine. We conclude that the ODC mediated route to putrescine plays an important role in determining the normal nicotine:anatabine profile in N. tabacum and is essential in allowing N. tabacum to increase nicotine levels in response to wound-associated stress.