Temperature remarkably affects the growth and metabolism of plants. Tobacco is an important cash crop, and the long-term effects of different growth temperatures (18.5, 23.5 and 28.5 °C, daily average) on growth, development and plastid pigments metabolism of tobacco plants were investigated in this study. Compared with tobacco plants grown under 23.5 °C, treatments with 18.5 and 28.5 °C inhibited the expansion of leaves. The contents of superoxide anion (O 2 ·− ), hydrogen peroxide (H2O2) and malonaldehyde (MDA) in the leaves were significantly increased under 28.5 °C from 0 to 60 days, which in turn accelerated the flowering and senescence of tobacco plants. By contrast, the treatment with 18.5 °C remarkably decreased O 2 .− , H2O2 and MDA, and delayed the flowering and senescence. Furthermore, treatment with 18.5 °C significantly up-regulated the expression of glutamyl-tRNA reductase (Glu-TR) and magnesium chelatase (MgCH), and down-regulated the ferri chelatase (FeCH), protochlorophyllide oxidoreductase, chlorophyllase (CHLase), phaeophorbide a monooxygenase (PaO) and phytoene synthase (PSY), which further promoted the accumulation of chlorophyll (Chls) and reduced the carotenoids (Cars) in leaves. On the contrary, exposing to 28.5 °C remarkably down-regulated the Glu-TR and MgCH, and up-regulated the FeCH, CHLase, PaO and PSY, which in turn decreased the Chls and increased the Cars in tobacco leaves. As compared with the plants grown under 23.5 °C, lower (18.5 °C) and higher (28.5 °C) growth temperature inhibited the growth of tobacco plants. In general, treatment with 28.5 °C accelerated the flowering and senescence of tobacco plants by enhancing the accumulation of O 2 .− and H2O2 in leaves, while exposing to 18.5 °C had the opposite effects. Treatment with 18.5 °C increased the content of Chls and reduced the Cars in leaves. In contrast, Treatment with 28.5 °C decreased the Chls and increased the Cars. Moreover, both O 2 .− and H2O2 took part in the breakdown of Chls in tobacco leaves to some extent. The results suggest that growth temperature could regulate growth, development, and plastid pigments metabolism, and 23.5 °C could be an optimal temperature for growth, development and metabolism of plastid pigments of tobacco plants under the experimental conditions.

Tobacco alkaloids (e.g., nicotine) and their metabolized tobacco-specific nitrosamines (TSNAs) are very important compounds for tobacco quality and safety. A simple and specific liquid chromatography–tandem mass spectrometry method was developed for the simultaneous determination of eight tobacco alkaloids and their related four TSNAs in tobacco leaves. The milled tobacco was extracted using 0.1 mol/L ammonium acetate solution and purified using methanol. Mass spectrometry parameters including declustering potential and collision energy were optimized to ensure that both the TSNAs and the tobacco alkaloids have suitable responses. Recoveries for accuracy were in the range of 80.2–105.2%. Intra-day and inter-day repeatability were in the range of 1.7–12.1% and 6.4–18.7%, respectively. Limit of detection and limit of quantitation were estimated in the range of 6 ng/g–45 μg/g and 24 ng/g–90 μg/g, respectively. The established method was applied to investigate the distribution of tobacco alkaloids and TSNAs in four kinds of tobacco. The result showed that the burley and the flue-cured have the highest (0.00047%) and the lowest (0.000024%) percentage of transformation from alkaloids to TSNAs, respectively. Thus, this method can be used for a wide range of samples.

Eleven consecutive N′-n-acylnornicotines from cherry-red tobacco were structurally elucidated and quantitively analyzed using chromatography and mass spectrometry. All of these N′-n-acylnornicotines are first reported in cherry-red tobacco, whereas N′-propionylnornicotine, N′-n-valerylnornicotine, N′-n-nonanoylnornicotine and N′-n-undecanoylnornicotine are first reported in natural products. The concentration distribution of the identified N′-n-acylnornicotines was studied and it was found that N′-n-octanoylnornicotine and N′-n-hexanoylnornicotine showed the highest concentration, accounting for 94% of the detected N′-n-acylnornicotines. The cherry-red color density of the related tobacco leaves was found to be positively correlated with the concentration of the N′-n-acylnornicotines, whereas the ultraviolet–visible spectra of the N′-n-acylnornicotines showed no absorption larger than 300 nm, indicating the discovered compounds are not responsible for the cherry-red color appearance.

Introduction: Cherry-red tobacco is a flue-cured variant that possesses a distinctive “sticky rice” flavor, which is highly valued by the tobacco industry. However, the value of cherryred tobacco is dubious due to the possible health risks associated with tobacco-specific nitrosamines (TSNAs). Objective: This study aimed to investigate the chemical origin of the “sticky rice” flavor and to assess the potential health hazards of TSNAs. Method: An optimized untargeted analysis with gas chromatography-mass spectrometry and a targeted analysis with liquid chromatography-tandem mass spectrometry were conducted. Result: Over one hundred compounds were identified and quantified. Cherry-red tobacco and the normal control showed significant differences in forty-three of these compounds. Pyridine alkaloids and their derivatives constituted the main difference. Nornicotine, a demethylated product of nicotine in cherry-red tobacco, was confirmed to be pyrolyzed to 3-ethylpyridine, 3-methylpyridine, and other homologues, and transferred to the smoke during smoking. The smoke of cherry-red tobacco was found to contain much higher levels of N’-nitrosonornicotine, a TSNA derived from nornicotine, than that of normal flue-cured tobacco, while the levels of the other detected TSNAs were lower. The two types of tobacco had similar total amounts of the four TSNAs. Conclusion: The pyrolysis of nornicotine into 3-ethylpyridine and its homologues during smoking may be the main cause of the “sticky rice” flavor of cherry-red tobacco. The level of TSNAs does not reflect the difference in health risk between cherry-red tobacco and the control.