Stimulation of Nicotine Demethylation by NaHCO₃ Treatment Using Greenhouse-Grown Burley Tobacco

Abstract

Experiments were conducted in tobacco (Nicotiana tabacum L.) to investigate the effect of sodium bicarbonate (NaHCO(3)) on the conversion of nicotine to nornicotine, a secondary alkaloid that can form the tobacco-specific nitrosamine N-nitrosonornicotine (NNN). The results showed that, under optimum conditions, NaHCO(3) stimulated nicotine conversion in converter plants to the maximum level predetermined by the genetic background. The conversion level in NaHCO(3)-treated leaves was 2-3 times higher than that in control leaves. For young seedlings the optimum concentration of sodium bicarbonate was 0.8% aqueous solution, and for adult plants the optimum concentration was 1%. Lower concentrations resulted in partial stimulation, whereas higher concentration damaged leaf tissue and resulted in a lower conversion level. Studies with different temperatures (from 22 to 43 degrees C) showed that 37 degrees C was optimal. This temperature allowed the least amount of time, 2-3 days for mature leaves and 4-6 days for green leaves, for the major converters to reach >95% of nicotine conversion. An examination of leaves from different growth stages and stalk positions showed that the amount of time needed for conversion was longer for young leaves and shorter for mature leaves. Treatment of leaves with NaHCO(3) affords a rapid and convenient means of identifying and removing nornicotine converter plants during growth in the greenhouse or field.

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase

“…For the microarray studies, individual leaves were excised, and their petioles were inserted into a solution of 0.1% ethephon or 1% sodium bicarbonate for 5-7 h, treatments that have been shown to accelerate the metabolism of nicotine to nornicotine (16,17). Treated leaves were subsequently placed in small plastic storage bags after being lightly sprayed with water (to maintain high humidity) and cured for 3 days at 30°C in the dark.…”

“…RNA immobilization, probe labeling, and signal detection were carried out by using the digoxigenin nucleic acid labeling and detection kits according to the manufacturer's instructions (Roche Applied Science). Alkaloid analysis of the sampled leaf material was performed as described (17).…”

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

“…Transgenic lines with a single transgene locus (segregation ratio of 3:1) were used in subsequent analyses to minimize gene dosage effects (Supplemental Table S1). Previously harvested T0 leaves of 12 OX lines (labeled as OX-1 to OX-12) and 10 KD lines (labeled as KD-1 to KD-10) were selected and subjected to nicotine and nornicotine analyses as described previously (Shi et al, 2003).We observed 3-to 4-fold higher total alkaloid contents in KD and their vector control lines compared with OX and their vector control lines (Supplemental Table S2). This is because the plants were grown at different times of the year: the KD plants were grown from April to July, while the OX plants were grown from November to February.…”

“…Nicotine and nornicotine contents were measured twice in triplicate assays following the method described by Shi et al (2003) using a Perkin-Elmer Autosystem XL Gas Chromatograph. NCR was calculated as: nornicotine/ (nicotine + nornicotine) 3 100%.…”

“…Transgenic lines with a single transgene locus (segregation ratio of 3:1) were used in subsequent analyses to minimize gene dosage effects (Supplemental Table S1). Previously harvested T0 leaves of 12 OX lines (labeled as OX-1 to OX-12) and 10 KD lines (labeled as KD-1 to KD-10) were selected and subjected to nicotine and nornicotine analyses as described previously (Shi et al, 2003).…”