Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.).

BACKGROUND: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. RESULTS: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. CONCLUSION: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

CLCNt2 Mediates Nitrate Content in Tobacco Leaf, Impacting the Production of Tobacco-Specific Nitrosamines in Cured Leaves.

Nitrate accumulation in tobacco (Nicotiana tabacum L.) leaf, particularly in the burley (BU) type, is a reservoir for the generation of nitrosating agents responsible for the formation of tobacco-specific nitrosamines (TSNAs). TSNAs are mainly produced via the nitrosation of alkaloids occurring during the curing of tobacco leaves. Additional formation of TSNAs may also occur during tobacco storage, leaf processing and in some circumstances via pyrosynthesis during combustion. Two TSNA species, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) are found in the tobacco products and have been documented to be animal carcinogens. A previous study showed that decreasing the accumulation of nitrate in tobacco leaf via the overexpression of a deregulated form of nitrate reductase is efficient to reduce the production of TSNAs. We pursue in finding another molecular genetic target to lower nitrate in BU tobacco. Suppressing expression or knocking-out CLCNt2 has a direct impact on leaf nitrate and TSNA reduction in cured leaves without altering biomass. This study provides now a straight path toward the development of new commercial tobacco varieties with reduced TSNA levels by breeding of variants deficient in active CLCNt2 copies.

Jasmonate Precursor Biosynthetic Enzymes LOX3 and LOX4 Control Wound-Response Growth Restriction.

Wound-response plant growth restriction requires the synthesis of potent mediators called jasmonates (JAs). Four 13-lipoxygenases (13-LOXs) produce JA precursors in Arabidopsis (Arabidopsis thaliana) leaves, but the 13-LOXs responsible for growth restriction have not yet been identified. Through loss-of-function genetic analyses, we identified LOX3 and LOX4 as the principal 13-LOXs responsible for vegetative growth restriction after repetitive wounding. Additional genetic studies were carried out in the gain-of-function fatty acid oxygenation 2 (fou2) mutant that, even when undamaged, shows JA-dependent leaf growth restriction. The fou2 lox3 lox4 triple mutant suppressed the fou2 JA-dependent growth phenotype, confirming that LOX3 and LOX4 function in leaf growth restriction. The fou2 mutation affects the TWO PORE CHANNEL1 (TPC1) ion channel. Additional genetic approaches based on this gene were used to further investigate LOX3 function in relation to leaf growth. To activate LOX3-dependent JA production in unwounded plants, we employed hyperactive TPC1 variants. Expression of the TPC1ΔCa i variant in phloem companion cells caused strongly reduced rosette growth in the absence of wounding. Summarizing, in parallel to their established roles in male reproductive development in Arabidopsis, LOX3 and LOX4 control leaf growth rates after wounding. The process of wound-response growth restriction can be recapitulated in unwounded plants when the LOX3 pathway is activated genetically using a hyperactive vacuolar cation channel.

Asparagine Synthesis During Tobacco Leaf Curing.

Senescence is a genetically controlled mechanism that modifies leaf chemistry. This involves significant changes in the accumulation of carbon- and nitrogen-containing compounds, including asparagine through the activity of asparagine synthetases. These enzymes are required for nitrogen re-assimilation and remobilization in plants; however, their mechanisms are not fully understood. Here, we report how leaf curing-a senescence-induced process that allows tobacco leaves to dry out-modifies the asparagine metabolism. We show that leaf curing strongly alters the concentration of the four main amino acids, asparagine, glutamine, aspartate, and glutamate. We demonstrate that detached tobacco leaf or stalk curing has a different impact on the expression of asparagine synthetase genes and accumulation of asparagine. Additionally, we characterize the main asparagine synthetases involved in the production of asparagine during curing. The expression of ASN1 and ASN5 genes is upregulated during curing. The ASN1-RNAi and ASN5-RNAi tobacco plant lines display significant alterations in the accumulation of asparagine, glutamine, and aspartate relative to wild-type plants. These results support the idea that ASN1 and ASN5 are key regulators of asparagine metabolism during leaf curing.