Genome-wide analysis of long noncoding RNAs in response to salt stress in Nicotiana tabacum.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to play important roles in the response of plants to various abiotic stresses, including drought, heat and salt stress. However, the identification and characterization of genome-wide salt-responsive lncRNAs in tobacco (Nicotiana tabacum L.) have been limited. Therefore, this study aimed to identify tobacco lncRNAs in roots and leaves in response to different durations of salt stress treatment. RESULTS: A total of 5,831 lncRNAs were discovered, with 2,428 classified as differentially expressed lncRNAs (DElncRNAs) in response to salt stress. Among these, only 214 DElncRNAs were shared between the 2,147 DElncRNAs in roots and the 495 DElncRNAs in leaves. KEGG pathway enrichment analysis revealed that these DElncRNAs were primarily associated with pathways involved in starch and sucrose metabolism in roots and cysteine and methionine metabolism pathway in leaves. Furthermore, weighted gene co-expression network analysis (WGCNA) identified 15 co-expression modules, with four modules strongly linked to salt stress across different treatment durations (MEsalmon, MElightgreen, MEgreenyellow and MEdarkred). Additionally, an lncRNA-miRNA-mRNA network was constructed, incorporating several known salt-associated miRNAs such as miR156, miR169 and miR396. CONCLUSIONS: This study enhances our understanding of the role of lncRNAs in the response of tobacco to salt stress. It provides valuable information on co-expression networks of lncRNA and mRNAs, as well as networks of lncRNAs-miRNAs-mRNAs. These findings identify important candidate lncRNAs that warrant further investigation in the study of plant-environment interactions.

Rational design of geranylgeranyl diphosphate synthase enhances carotenoid production and improves photosynthetic efficiency in Nicotiana tabacum.

Restricted genetic diversity can supply only a limited number of elite genes for modern plant cultivation and transgenesis. In this study, we demonstrate that rational design enables the engineering of geranylgeranyl diphosphate synthase (NtGGPPS), an enzyme of the methylerythritol phosphate pathway (MEP) in the model plant Nicotiana tabacum. As the crucial bottleneck in carotenoid biosynthesis, NtGGPPS1 interacts with phytoene synthase (NtPSY1) to channel GGPP into the production of carotenoids. Loss of this enzyme in the ntggpps1 mutant leads to decreased carotenoid accumulation. With the aim of enhancing NtGGPPS1 activity, we undertook structure-guided rational redesign of its substrate binding pocket in combination with sequence alignment. The activity of the designed NtGGPPS1 (a pentuple mutant of five sites V154A/I161L/F218Y/I209S/V233E, d-NtGGPPS1) was measured by a high-throughput colorimetric assay. d-NtGGPPS1 exhibited significantly higher conversion of IPP and each co-substrate (DMAPP ~1995.5-fold, GPP ~25.9-fold, and FPP ~16.7-fold) for GGPP synthesis compared with wild-type NtGGPPS1. Importantly, the transient and stable expression of d-NtGGPPS1 in the ntggpps1 mutant increased carotenoid levels in leaves, improved photosynthetic efficiency, and increased biomass relative to NtGGPPS1. These findings provide a firm basis for the engineering of GGPPS and will facilitate the development of quality and yield traits. Our results open the door for the structure-guided rational design of elite genes in higher plants.

Genome-wide identification and characterization of NPF family reveals NtNPF6.13 involving in salt stress in Nicotiana tabacum.

Proteins of the Nitrate Transporter 1/Peptide Transporter (NPF) family transport a diverse variety of substrates, such as nitrate, peptides, hormones and chloride. In this study, a systematic analysis of the tobacco (Nicotiana tabacum) NPF family was performed in the cultivated 'K326'. In total, 143 NtNPF genes were identified and phylogenetically classified into eight subfamilies, NPF1 to NPF8, based on the classification of NPF families in other plant species. The chromosomal locations and structures of the NtNPF genes were analyzed. The expression profiles of NtNPF genes under NaCl stress were analyzed to screen the possible NPF genes involving in chloride regulation in tobacco. Most NtNPF6 genes responded to salt stress in the roots and leaves. The expression of NtNPF6.13 was significantly down-regulated after salt stress for 12h. The chloride content was reduced in the roots of ntnpf6.13 mutant. These findings support the participation of NtNPF6.13 in chloride uptake. Several other NtNPF genes that play potential roles in chloride metabolism of tobacco require further study.

Functional characterization and comparison of lycopene epsilon-cyclase genes in Nicotiana tabacum.

BACKGROUND: Lycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants. RESULTS: Here, we used CRISPR/Cas9 to obtain Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants. CONCLUSION: In summary, the expression patterns and biological functions of the Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.

Reconstruction of the full-length transcriptome of cigar tobacco without a reference genome and characterization of anion channel/transporter transcripts.

BACKGROUND: Cigar wrapper leaves are the most important raw material of cigars. Studying the genomic information of cigar tobacco is conducive to improving cigar quality from the perspective of genetic breeding. However, no reference genome or full-length transcripts at the genome-wide scale have been reported for cigar tobacco. In particular, anion channels/transporters are of high interest for their potential application in regulating the chloride content of cigar tobacco growing on coastal lands, which usually results in relatively high Cl- accumulation, which is unfavorable. Here, the PacBio platform and NGS technology were combined to generate a full-length transcriptome of cigar tobacco used for cigar wrappers. RESULTS: High-quality RNA isolated from the roots, leaves and stems of cigar tobacco were subjected to both the PacBio platform and NGS. From PacBio, a total of 11,652,432 subreads (19-Gb) were generated, with an average read length of 1,608 bp. After corrections were performed in conjunction with the NGS reads, we ultimately identified 1,695,064 open reading frames including 21,486 full-length ORFs and 7,342 genes encoding transcription factors from 55 TF families, together with 2,230 genes encoding long non-coding RNAs. Members of gene families related to anion channels/transporters, including members of the SLAC and CLC families, were identified and characterized. CONCLUSIONS: The full-length transcriptome of cigar tobacco was obtained, annotated, and analyzed, providing a valuable genetic resource for future studies in cigar tobacco.

Characterization and functional analysis of phytoene synthase gene family in tobacco.

BACKGROUND: Carotenoids play important roles in photosynthesis, hormone signaling, and secondary metabolism. Phytoene synthase (PSY) catalyzes the first step of the carotenoid biosynthetic pathway. In this study, we aimed to characterize the PSY genes in tobacco and analyze their function. RESULTS: In this study, we identified three groups of PSY genes, namely PSY1, PSY2, and PSY3, in four Nicotiana species; phylogenetic analysis indicated that these genes shared a high similarity with those in tomato but not with those in monocots such as rice and maize. The expression levels of PSY1 and PSY2 were observed to be highest in leaves compared to other tissues, and they could be elevated by treatment with certain phytohormones and exposure to strong light. No PSY3 expression was detected under these conditions. We constructed virus-induced PSY1 and PSY2 silencing in tobacco and found that the newly emerged leaves in these plants were characterized by severe bleaching and markedly decreased carotenoid and chlorophyll content. Thylakoid membrane protein complex levels in the gene-silenced plants were also less than those in the control plants. The chlorophyll fluorescence parameters such as Fv/Fm, ΦPSII, qP, and NPQ, which reflect photosynthetic system activities, of the gene-silenced plants were also significantly decreased. We further performed RNA-Seq and metabonomics analysis between gene-silenced tobacco and control plants. RNA-Seq results showed that abiotic stress, isoprenoid compounds, and amino acid catabolic processes were upregulated, whereas the biosynthesis of cell wall components was downregulated. Metabolic analysis results were consistent with the RNA-Seq. We also found the downstream genes in carotenoid biosynthesis pathways were upregulated, and putative transcription factors that regulate carotenoid biosynthesis were identified. CONCLUSIONS: Our results suggest that PSY can regulate carotenoid contents not only by controlling the first biosynthesis step but also by exerting effects on the expression of downstream genes, which would thereby affect photosynthetic activity. Meanwhile, PSY may affect other processes such as amino acid catabolism and cell wall organization. The information we report here may aid further research on PSY genes and carotenoid biosynthesis.

Integrating transcriptome and metabolome reveals molecular networks involved in genetic and environmental variation in tobacco.

Tobacco (Nicotiana tabacum) is one of the most widely cultivated commercial non-food crops with significant social and economic impacts. Here we profiled transcriptome and metabolome from 54 tobacco samples (2-3 replicates; n = 151 in total) collected from three varieties (i.e. genetic factor), three locations (i.e. environmental factor), and six developmental stages (i.e. developmental process). We identified 3,405 differentially expressed (DE) genes (DEGs) and 371 DE metabolites, respectively. We used quantitative real-time PCR to validate 20 DEGs, and confirmed 18/20 (90%) DEGs between three locations and 16/20 (80%) with the same trend across developmental stages. We then constructed nine co-expression gene modules and four co-expression metabolite modules , and defined seven de novo regulatory networks, including nicotine- and carotenoid-related regulatory networks. A novel two-way Pearson correlation approach was further proposed to integrate co-expression gene and metabolite modules to identify joint gene-metabolite relations. Finally, we further integrated DE and network results to prioritize genes by its functional importance and identified a top-ranked novel gene, LOC107773232, as a potential regulator involved in the carotenoid metabolism pathway. Thus, the results and systems-biology approaches provide a new avenue to understand the molecular mechanisms underlying complex genetic and environmental perturbations in tobacco.

Four-week administration of nicotinemoderately impacts blood metabolic profile and gut microbiota in a diet-dependent manner.

As the primary active component in tobacco, nicotine affects many aspects of human metabolism. Diet and gut microbiota are key factors that profoundly influence human lipid and glucose metabolism. However, the diet-based differential impacts of nicotine on blood lipid and glucose levels as well as on the gut microbiota are still largely unknown. Here we show that 4-week oral administration of nicotine (2 mg/kg) resulted in bodyweight and fat decrease in both normal-chow (NCD)- and high-fat diet (HFD)-fed mice. But nicotine showed little influence on the plasma levels of lipids, glucose and inflammatory cytokines in NCD-fed mice but moderately deteriorated these parameters in HFD-fed ones. 16S sequencing showed that nicotine perturbed bacterial diversity and community composition of gut microbiota more pronouncedly in HFD mice. At genus level, nicotine dramatically increased Ruminococcaceae UCG-009 in HFD condition but not in NCD feeding. Interestingly, co-treatment with antibiotics (ampicillin + norfloxacin) substantially abolished the lipid-enhancing effect of nicotine in HFD-fed mice, suggesting an important role of gut microbes in the lipid-modulatory effect of nicotine. Together, our results indicate that the harmful effects of nicotine on metabolism and systemic inflammation are diet-dependent. Chronic exposure to nicotine may alter the gut microbiota especially in HFD-fed animals.

Identification and analysis of the chloride channel gene family members in tobacco (Nicotiana tabacum).

The chloride channel (CLC) protein family, which includes both chloride (Cl-) channels and chloride/proton (Cl-/H+) antiporters, is present in all domains of life, from prokaryotes to eukaryotes. However, there are no reported studies about this gene family in tobacco, an economically important global crop plant. In this study, we identified seventeen CLC genes in the genome of Nicotiana tabacum. A multiple sequence alignment showed that all of the predicted proteins shared a high sequence similarity and had a highly conserved GKxGPxxH motif. A gene structure analysis revealed that the NtCLC genes had highly divergent intron-exon patterns. A phylogenetic and conserved motif analysis revealed that the NtCLC family was divided into two clades, in a manner similar to other plants. We also evaluated the expression patterns of these NtCLC genes in different tissues and in plants treated with salt stress. The NtCLC genes had highly variable expression patterns, for example, the largely stem- and bud-specific expression patterns of NtCLC6 and NtCLC8, respectively. Salt stress treatment (300 mM NaCl) induced the expression of NtCLC2, NtCLC3, and NtCLC12, suggesting that these genes might play a role in tobacco responses to salt stress. Furthermore, the concentration of Cl- in the NtCLC2- and NtCLC13-silenced plants showed an obvious lower and higher level, respectively, than the control plants. Thus, we indicated that NtCLC2 or NtCLC13 might play an important role in chloride transport or metabolism in tobacco. Together, these findings establish an empirical foundation for the further functional characterization of the NtCLC genes in tobacco.

NtRLK5, a novel RLK-like protein kinase from Nitotiana tobacum, positively regulates drought tolerance in transgenic Arabidopsis.

Receptor-like protein kinase (RLKs) plays pivotal roles in plant growth and development as well as stress responses. However, little is known about the function of RLKs in Nitotiana tobacum. In the present study, we present data on NtRLK5, a novel RLK-like gene isolated from Hongda (Nitotiana tobacum L.). Expression profile analysis revealed that NtRLK5 was strongly induced by drought and salt stresses. Transient expression of NtRLK5-GFP fusion protein in protoplast showed that NtRLK5 was localized to plasma membrane. Overexpression of NtRLK5 conferred enhanced drought tolerance in transgenic Arabidopsis plants, which was attributed to not only the lower malondialdehyde (MDA) and H2O2 contents, but also the higher antioxidant enzymes activities. Moreover, the expression of several antioxidation- and stress-related genes was also significantly up-regulated in NtRLK5 transgenic plants under drought condition. Taken together, the results suggest that NtRLK5 functions as a positive regulator in drought tolerance.

A simple and cost-effective method for screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

BACKGROUND: The CRISPR/Cas9 system is being used for genome editing purposes by many research groups in multiple plant species. Traditional sequencing methods to identify homozygous mutants are time-consuming, laborious and expensive. RESULTS: We have developed a method to screen CRISPR/Cas9-induced mutants through Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR). The MSBSP-PCR method was successfully used to identify homozygous/biallelic mutants in Nicotiana tabacum and Arabidopsis thaliana, and we speculate that it can be used for the identification of CRISPR/Cas9-induced mutants in other plant species. Compared to traditional sequencing methods, MSBSP-PCR is simpler, faster and cheaper. CONCLUSIONS: The MSBSP-PCR method is simple to implement and can save time and cost in the screening of CRISPR/Cas9-induced homozygous/biallelic mutants.

Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco.

BACKGROUND: Drought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequencing on tobacco leaf samples from the following three treatments: untreated-control (CL), drought stress (DL), and re-watering (WL). RESULTS: In total, we identified 798 differentially expressed genes (DEGs) between the DL and CL (DL vs. CL) treatments and identified 571 DEGs between the WL and DL (WL vs. DL) treatments. Further analysis revealed 443 overlapping DEGs between the DL vs. CL and WL vs. DL comparisons, and, strikingly, all of these genes exhibited opposing expression trends between these two comparisons, strongly suggesting that these overlapping DEGs are somehow involved in the responses of tobacco leaves to drought stress. Functional annotation analysis showed significant up-regulation of genes annotated to be involved in responses to stimulus and stress, (e.g., late embryogenesis abundant proteins and heat-shock proteins) antioxidant defense (e.g., peroxidases and glutathione S-transferases), down regulation of genes related to the cell cycle pathway, and photosynthesis processes. We also found 69 and 56 transcription factors (TFs) among the DEGs in, respectively, the DL vs. CL and the WL vs. DL comparisons. In addition, small RNA sequencing revealed 63 known microRNAs (miRNA) from 32 families and 368 novel miRNA candidates in tobacco. We also found that five known miRNA families (miR398, miR390, miR162, miR166, and miR168) showed differential regulation under drought conditions. Analysis to identify negative correlations between the differentially expressed miRNAs (DEMs) and DEGs revealed 92 mRNA-miRNA interactions between CL and DL plants, and 32 mRNA-miRNA interactions between DL and WL plants. CONCLUSIONS: This study provides a global view of the transcriptional and the post-transcriptional responses of tobacco under drought stress and re-watering conditions. Our results establish an empirical foundation that should prove valuable for further investigations into the molecular mechanisms through which tobacco, and plants more generally, respond to drought stress at multiple molecular genetic levels.

Cloning of the Lycopene ß-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance.

Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene ß cyclase (ß-LCY) functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a ß-LCY gene from Nicotiana tabacum, designated as Ntß-LCY1, was cloned and functionally characterized. Robust expression of Ntß-LCY1 was found in leaves, and Ntß-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntß-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntß-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntß-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

High levels of plasma selenium are associated with metabolic syndrome and elevated fasting plasma glucose in a Chinese population: A case-control study.

BACKGROUND: Selenium is important for human health and involved in various metabolic processes. Deficiency of selenium associates with increased risk for cancer and cardiovascular diseases. There has been an increase use of selenium supplements for the treatment of autoimmune thyroid conditions. However, the potential biological effects of selenium overload arouse the public concern. The aim of this study was to investigate the associations of plasma selenium concentrations of adults with metabolic syndrome (MS) in Chinese population. METHODS: A matched case-control study including 204 metabolic syndrome patients and 204 healthy controls was conducted in 2012. The MS cases were defined according to the criteria of Chinese Diabetes Society (CDS). Healthy controls without abnormality of metabolic components were matched with cases in age, gender and region. Plasma concentrations of selenium were determined by graphite furnace atomic absorption spectrometry (GFAAS). Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein cholesterol (LDL) were detected by automatic biochemical analyzer. RESULTS: The median levels of plasma selenium in MS group were 146.3 (107.3-199.4)µg/L, which were significantly higher than that in the control group (127.4: 95.7-176.0)µg/L; Plasma levels of selenium were related to the risk of MS in dose-response manner. Risk of MS was significantly higher in subjects with plasma selenium in the highest tertile (T3: ≥176.0µg/L) compared to those in the lowest tertile (T1: <95.7µg/L) [odds ratio (OR)=2.416 (95% CI: 1.289-4.526)]. The plasma levels of selenium were positively correlated with fasting plasma glucose (FPG) (rs=0.268, P<0.001). Plasma selenium at the median (T2: 95.7-176.0µg/L) or upper tertile (T3: ≥176.0µg/L) was associated with increased risk of elevated FPG (defined by FPG≥6.1mmol/L) as compared with the lowest tertile (T1: ≤95.7µg/L) [T2 vs. T1, OR=3.487 (1.738-6.996); T3 vs. T1, OR=6.245 (3.005-12.981)]. CONCLUSIONS: Higher levels of plasma selenium might increase the risk of metabolic syndrome and elevated fasting plasma glucose. Selenium supplements should be used with prudence for CVD and cancer prevention.

Molecular cloning and functional characterization of the lycopene ε-cyclase gene via virus-induced gene silencing and its expression pattern in Nicotiana tabacum.

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of ß-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of ß-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Aphanamixins A-F, acyclic diterpenoids from the stem bark of Aphanamixis polystachya.

Six new acyclic diterpenoids named Aphanamixins A-F (1-6), together with two known compounds of nemoralisin and nemoralisin C, were isolated from the stem bark of Aphanamixis polystachya (WALL) J. N. BARKER. Their structures were established through a comprehensive analysis of NMR spectroscopic data and high resolution mass spectrometric data. The absolute configurations of carbon stereocenters were determined by means of auxiliary chiral α-methoxy-α-(trifluoromethyl)phenylacetic acid (MTPA) derivatives and circular dichroism (CD), respectively. All the new isolates were tested for their antiproliferative activity against HepG2, AGS, MCF-7, and A-549 cancer cell lines and they exhibited weak cytotoxicities (IC50>10 µM). Moreover, we highlighted that the six new diterpenoids characterized by acyclic skeleton was rarely seen in nature.

Genetic variation in alkaloid accumulation in leaves of Nicotiana.

Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R(2)=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.

Modulation of lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes by sophoricoside.

Sophoricoside, an isoflavone glycoside isolated from Sophora japonica (Leguminosae), has been widely reported as an immunomodulator. In this study, the effects of sophoricoside on lipogenesis and glucose consumption in HepG2 cells and C2C12 myotubes were investigated. Treatment with sophoricoside at concentrations of 1-10 µM inhibited lipid accumulation in HepG2 cells in a dose-dependent manner. At the same concentration range, no effect on cell viability was observed in the MTT assay. Inhibition of lipogenesis was associated with the downregulation of SREBP-1a, SREBP-1c, SREBP-2 and their downstream target genes (FAS, ACC, HMGR) as revealed by realtime quantitative PCR. The lipid-lowering effect was mediated via the phosphorylation of AMPK. Further investigation of the activities of this isoflavone showed that sophoricoside has the capability to increase glucose uptake by C2C12 myotubes. It also effectively inhibited the activities of α-glucosidase and α-amylase in vitro and remarkably lowered postprandial hyperglycaemia in starch-loaded C57BL6/J mice. These results suggest that sophoricoside is an effective regulator of lipogenesis and glucose consumption and may find utility in the treatment of obesity and type 2 diabetes.

Investigation of the relationship between the metabolic profile of tobacco leaves in different planting regions and climate factors using a pseudotargeted method based on gas chromatography/mass spectrometry.

An improved pseudotargeted method using gas chromatography/mass spectrometry (GC/MS) was developed to investigate the metabolic profile of tobacco leaves from three planting regions (Yunnan, Guizhou, and Henan provinces). The analytical characteristics of the method with regard to reproducibility, precision, linearity, and stability were satisfactory for metabolic profiling study. Partial least-squares-discriminant analysis and hierarchical cluster analysis demonstrated that the metabolic profiles of tobacco from the Yunnan and Guizhou regions were different from that from the Henan province. The amino acid (e.g., phenylalanine, leucine, and tyrosine) and carbohydrate (e.g., fructose, trehalose, and sucrose) contents were the highest in Henan tobacco. The highest contents of organic acids (e.g., isocitrate, citrate, and fumarate) of the TCA cycle and antioxidants (e.g., quinate, chlorogenic acid, and ascorbate) were found in Guizhou tobacco. The correlation coefficients between metabolite content and climate factors (rainfall, sunshine, and temperature) demonstrated that drought facilitated the accumulation of sugars and amino acids. The content of TCA cycle intermediates could be influenced by multiple climate factors. This study demonstrates that the pseudotargeted method with GC/MS is suitable for the investigation of the metabolic profiling of tobacco leaves and the assessment of differential metabolite levels related to the growing regions.