Wenjie Pan

Determination of carbohydrates in tobacco by pressurized liquid extraction combined with a novel ultrasound-assisted dispersive liquid–liquid microextraction method

A novel derivatization-ultrasonic assisted-dispersive liquid-liquid microextraction (UA-DLLME) method for the simultaneous determination of 11 main carbohydrates in tobacco has been developed. The combined method involves pressurized liquid extraction (PLE), derivatization, and UA-DLLME, followed by the analysis of the main carbohydrates with a gas chromatography-flame ionization detector (GC-FID). First, the PLE conditions were optimized using a univariate approach. Then, the derivatization methods were properly compared and optimized. The aldononitrile acetate method combined with the O-methoxyoxime-trimethylsilyl method was used for derivatization. Finally, the critical variables affecting the UA-DLLME extraction efficiency were searched using fractional factorial design (FFD) and further optimized using Doehlert design (DD) of the response surface methodology. The optimum conditions were found to be 44 μL for CHCl3, 2.3 mL for H2O, 11% w/v for NaCl, 5 min for the extraction time and 5 min for the centrifugation time. Under the optimized experimental conditions, the detection limit of the method (LODs) and linear correlation coefficient were found to be in the range of 0.06-0.90 μg mL(-1) and 0.9987-0.9999. The proposed method was successfully employed to analyze three flue-cured tobacco cultivars, among which the main carbohydrate concentrations were found to be very different.

RNA Sequencing Analysis Reveals Transcriptomic Variations in Tobacco (Nicotiana tabacum) Leaves Affected by Climate, Soil, and Tillage Factors

The growth and development of plants are sensitive to their surroundings. Although numerous studies have analyzed plant transcriptomic variation, few have quantified the effect of combinations of factors or identified factor-specific effects. In this study, we performed RNA sequencing (RNA-seq) analysis on tobacco leaves derived from 10 treatment combinations of three groups of ecological factors, i.e., climate factors (CFs), soil factors (SFs), and tillage factors (TFs). We detected 4980, 2916, and 1605 differentially expressed genes (DEGs) that were affected by CFs, SFs, and TFs, which included 2703, 768, and 507 specific and 703 common DEGs (simultaneously regulated by CFs, SFs, and TFs), respectively. GO and KEGG enrichment analyses showed that genes involved in abiotic stress responses and secondary metabolic pathways were overrepresented in the common and CF-specific DEGs. In addition, we noted enrichment in CF-specific DEGs related to the circadian rhythm, SF-specific DEGs involved in mineral nutrient absorption and transport, and SF- and TF-specific DEGs associated with photosynthesis. Based on these results, we propose a model that explains how plants adapt to various ecological factors at the transcriptomic level. Additionally, the identified DEGs lay the foundation for future investigations of stress resistance, circadian rhythm and photosynthesis in tobacco.

Identification and quantitation of glycosidically bound aroma compounds in three tobacco types by gas chromatography–mass spectrometry

Glycosidically bound aroma compounds in three different types of tobacco were investigated. After isolation of extracts obtained by Amberlite XAD-2 adsorption and ethyl acetate elution, glycosides were analyzed after enzymatic hydrolysis by gas chromatography-mass spectrometry (GC-MS) or directly after trifluoroacetylated (TFA) derivatization by GC-MS in electron ionization (EI) and negative chemical ionization (NCI) mode. In total 21 bound aglycones were identified by β-glucosidase hydrolysis. These aglycones mainly consisted of C13-norisoprenoids, aromatic components and sesquiterpenoids. Additionally, with the aid of enzymatic hydrolysis, 15 β-d-glucopyranosides and 1 β-d-rutinoside were tentatively identified by TFA derivatization. TFA method was validated by repeatability and successfully employed to analyze different types of tobacco. Principal component analysis (PCA) was carried out on identified glycoside variables to visualize the difference between the tobacco types and the relationship between the glycoside variables and the tobacco types was established.

Enzymatic hydrolysis followed by gas chromatography-mass spectroscopy for determination of glycosides in tobacco and method optimization by response surface methodology

To evaluate the glycoconjugated aroma compounds of three cultivars of tobacco, an enzymatic hydrolysis method followed by gas chromatography-mass spectroscopy (GC-MS) was successfully established using a series optimization approach. In the first step, two hydrolytic enzymes were compared and β-glucosidase was chosen. Then, the critical variables that affect the concentration of aglycons were searched by Plackett–Burman design (PBD) and further optimized by central composite design (CCD) of response surface methodology. The optimum conditions were: 60 h for hydrolysis time, 36.8 °C for hydrolysis temperature, 5.6 for pH, 2.1 mL for solvent volume, 2 mg mL−1 for β-glucosidase concentration and 200 rpm for agitation speed. Under the optimized experimental conditions, this method gave a satisfactory precision and linearity with relative standard deviation (RSD) less than 20% and Pearson correlation coefficients greater than 0.99. Finally, the proposed method was successfully employed to analyze flue-cured tobacco (FCT), sun-cured tobacco (SCT) and oriental tobacco (OT). Most of the aglycons were significantly different in the three cultivars and the corresponding characteristic components were discussed in detail.

Comparative transcriptome analysis of tobacco (Nicotiana tabacum) leaves to identify aroma compound-related genes expressed in different cultivated regions.

To identify genes that are differentially expressed in tobacco in response to environmental changes and to decipher the mechanisms by which aromatic carotenoids are formed in tobacco, an Agilent Tobacco Gene Expression microarray was adapted for transcriptome comparison of tobacco leaves derived from three cultivated regions of China, Kaiyang (KY), Weining (WN) and Tianzhu (TZ). A total of 1,005 genes were differentially expressed between leaves derived from KY and TZ, 733 between KY and WN, and 517 between TZ and WN. Genes that were upregulated in leaves from WN and TZ tended to be involved in secondary metabolism pathways, and included several carotenoid pathway genes, e.g., NtPYS, NtPDS, and NtLCYE, whereas those that were down-regulated tended to be involved in the response to temperature and light. The expression of 10 differentially expressed genes (DEGs) was evaluated by real-time quantitative polymerase chain reaction (qRT-PCR) and found to be consistent with the microarray data. Gene Ontology and MapMan analyses indicate that the genes that were differentially expressed among the three cultivated regions were associated with the light reaction of photosystem II, response to stimuli, and secondary metabolism. High-performance liquid chromatography (HPLC) analysis showed that leaves derived from KY had the lowest levels of lutein, β-carotene, and neoxanthin, whereas the total carotenoid content in leaves from TZ was greatest, a finding that could well be explained by the expression patterns of DEGs in the carotenoid pathway. These results may help elucidate the molecular mechanisms underlying environmental adaptation and accumulation of aroma compounds in tobacco.

Low-temperature derivatization followed by vortex-assisted liquid-liquid microextraction for the analysis of polyamines in Nicotiana Tabacum.

Polyamines are ubiquitous polycationic molecules that play a key role in many biological processes such as nucleic acid metabolism, protein synthesis, cell growth, and nicotine synthesis precursors. This work describes a rapid, sensitive, convenient, green, and cost-effective method for the determination of polyamines in Nicotiana tabacum by ultra high performance liquid chromatography with photodiode array detection. The analytes were derivatized with 3,5-dinitrobenzoyl chloride at low temperature (about 4°C) and then extracted with vortex-assisted liquid-liquid microextraction. The experimental designs based on quarter-fractional factorial design and Doehlert design were used to screen and optimize the important factors in microextraction process. Under the optimal conditions, the method was linear over 0.05-8.00 μg/mL with an r(2) ≥ 0.992 and exhibited good repeatability and reproducibility less than 6.0 and 6.9%, respectively. The limit of detection ranged between 0.013 and 0.029 μg/g. The newly developed method was successfully employed to analyze different leaf samples of Nicotiana tabacum, among which the polyamines contents were found to be very different. Moreover, tyramine, 1,3-diaminopropane, homospermidine, and canavalmine were tentatively identified with the electrospray ionization quadrupole time-of-flight mass spectrometry. To our knowledge, this is the first report of identification of canavalmine in Nicotiana Tabacum.

Exogenous trehalose improves growth under limiting nitrogen through upregulation of nitrogen metabolism

BackgroundThe trehalose (Tre) pathway has strong effects on growth and development in plants through regulation of carbon metabolism. Altering either Tre or trehalose 6-phosphate (T6P) can improve growth and productivity of plants as observed under different water availability. As yet, there are no reports of the effects of modification of Tre orT6P on plant performance under limiting nutrition.ResultsHere we report that nitrogen (N) metabolism is positively affected by exogenous application of Tre in nitrogen-deficient growing conditions. Spraying foliage of tobacco (Nicotiana tabacum) with trehalose partially alleviated symptoms of nitrogen deficiency through upregulation of nitrate and ammonia assimilation and increasing activities of nitrate reductase (NR), glycolate oxidase (GO), glutamine synthetase (GS) and glutamine oxoglutarate aminotransferase (GOGAT) with concomitant changes in ammonium (NH4+) and nitrate (NO3−) concentrations, glutamine and amino acids. Chlorophyll and total nitrogen content of leaves and rates of photosynthesis were increased compared to nitrogen-deficient plants without applied Tre. Total plant biomass accumulation was also higher in Tre -fed nitrogen-deficient plants, with a smaller proportion of dry weight partitioned to roots, compared to nitrogen-deficient plants without applied Tre. Consistent with higher nitrogen assimilation and growth, Tre application reduced foliar starch. Minimal effects of Tre feeding were observed on nitrogen-sufficient plants.ConclusionsThe data show, for the first time, significant stimulatory effects of exogenous Tre on nitrogen metabolism and growth in plants growing under deficient nitrogen. Under such adverse conditions metabolism is regulated for survival rather than productivity. Application of Tre can alter this regulation towards maintenance of productive functions under low nitrogen. This has implications for considering approaches to modifying the Tre pathway for to improve crop nitrogen-use efficiency and production.

Analysis of nitrogenous organic compounds from mainstream cigarette smoke using low-temperature solvent extraction followed by comprehensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry.

A method involving comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry was developed and applied to the analysis of nitrogenous organic compounds present in mainstream cigarette smoke trapped on self-designed equipment. The samples were prepared using low-temperature solvent extraction under liquid nitrogen and analyzed by comprehensive two-dimensional gas chromatography with high-resolution time-of-flight mass spectrometry. Important experimental parameters, such as the type and volume of the extraction solvent and flow rate of smoking, were optimized to improve the analysis parameter. The results indicated that 180 mL of diethyl ether in the low-temperature solvent extraction apparatus system with a 4 mL/min smoke flow rate were the optimal conditions. Then, 85 nitrogenous organic compounds were identified and quantified using a mass spectral library search, accurate mass ion and N-rules of a molecular formula for nitrogen compounds. Finally, a comparison of the low temperature solvent extraction method and Cambridge filter pad method indicated that more peaks, a higher peak volume and better repeatability were obtained using the low-temperature solvent extraction method.

Effects of biochar on Cd and Pb mobility and microbial community composition in a calcareous soil planted with tobacco

An experiment was conducted with tobacco (Nicotiana tabacum L.) grown in a Cd- and Pb-contaminated calcareous soil amended with 0.0, 1.0, 2.5, and 5.0% (w/w) tobacco stalk biochar (BC). The BC amendment significantly increased organic matter, total C, N, P, and K contents of soil, and the C/N ratio. Bioavailable metal concentrations (DTPA extraction) decreased by increasing BC application rate. The 5.0% BC amendment significantly decreased the DTPA-extractable Cd and Pb by 10.4 and 13.6%, respectively. Correspondingly, the bioaccumulation and translocation factors of Cd and Pb also decreased by increasing the BC addition rates and this indicated that BC inhibited the uptake and transfer of both Cd and Pb by tobacco plants. Moreover, high-throughput sequencing revealed that BC increased Chao1 richness, Shannon’s diversity and Simpson’s diversity of bacterial communities of soil. The relative abundance and genera composition of Adhaeribacter, Rhodoplanes, Pseudoxanthomonas, and Candidatus Xiphinematobacter increased under BC treatments, while those of Kaistobacter, Lacibacter, and Pirellula decreased. Overall, BC increased soil nutrients (C, N, P, and K contents), enhanced bacterial diversity indexes and richness, and changed the bacterial community composition, which may all have contributed to reduce the mobility and bioavailability of both Cd and Pb in a calcareous soil.