The nicotine demethylase CYP82E4 is essential for the formation of red dapples on flue-cured leaves of cherry-red tobacco.

Common flue-cured tobacco (Nicotiana tabacum L.) primarily accumulates nicotine, and its flue-cured leaves exhibit a lemon appearance. In contrast, a spontaneous cherry-red variant (CR60) primarily accumulates nornicotine, accompanied by distinctive red dapples on the cured leaves. In this study, suppression of conversion of nicotine to nornicotine by genome editing resulted in decreased nornicotine and N-acyl nornicotines (NacNNs), and the subsequent disappearance of red dapples in CR60. Conversely, overexpression of CYP82E4 increased nornicotine and NacNNs accumulation, inducing a red dapple phenotype in common tobacco. Notably, nicotine conversion triggered significant alterations in leaf total sugars, alkaloids, and nitrogens. Metabolome analyses using 1352 identified compounds indicated nicotine conversion dramatically affected the entire metabolic network and induced unique metabolic responses across diverse genetic backgrounds. Further WGCNA analysis revealed that nicotine conversion caused substantial contents variation of alkaloids, flavonoids and amino acids and derivatives in cured leaves. Overall, this research provides valuable insights into the mechanisms underlying red dapple formation in cherry-red tobacco, elucidating profound influence of nicotine conversion on entire metabolic network.

A tonoplast-localized TPK-type K+ transporter (TPKa) regulates potassium accumulation in tobacco.

Potassium ion (K+) is one of the most essential nutrients for the growth and development of tobacco (Nicotiana tabacum L.), however, the molecular regulation of K+ concentration in tobacco remains unclear. In this study, a two-pore K (TPK) channel gene NtTPKa was cloned from tobacco, and NtTPKa protein contains the unique K+ selection motif GYGD and its transmembrane region primarily locates in the tonoplast membrane. The expression of NtTPKa gene was significantly increased under low-potassium stress conditions. The concentrations of K+ in tobacco were significantly increased in the NtTPKa RNA interference lines and CRISPR/Cas9 knockout mutants. In addition, the transport of K+ by NtTPKa was validated using patch clamp technique, and the results showed that NtTPKa channel protein exclusively transported K+ in a concentration-dependent manner. Together, our results strongly suggested that NtTPKa is a key gene in maintaining K+ homeostasis in tobacco, and it could provide a new genetic resource for increasing the concentration of K+ in tobacco.

Comparative transcriptome analysis reveals nicotine metabolism is a critical component for enhancing stress response intensity of innate immunity system in tobacco.

The pyridine alkaloid nicotine acts as one of best-studied plant resistant traits in tobacco. Previous research has shown that NtERF199 and NtERF189, acting as master regulators within the NIC1 and NIC2 locus, quantitatively contribute to nicotine accumulation levels in N. tabacum. Genome editing-created Nic1(Nterf199) and Nic2 (Nterf189) double mutant provides an ideal platform for precisely dissecting the defensive role of nicotine and the connection between the nicotine biosynthetic pathway with other putative metabolic networks. Taking this advantage, we performed a comparative transcriptomic analysis to reevaluate the potential physiological and metabolic changes in response to nicotine synthesis defect by comparing the nic1nic2 and NIC1NIC2 plants. Our findings revealed that nicotine reduction could systematically diminishes the expression intensities of genes associated with stimulus perception, signal transduction and regulation, as well as secondary metabolic flux. Consequently, this global expression reduction might compromise tobacco adaptions to environmental fitness, herbivore resistances, and plant growth and development. The up-regulation of a novel set of stress-responsive and metabolic pathway genes might signify a newly established metabolic reprogramming to tradeoff the detrimental effect of nicotine loss. These results offer additional compelling evidence regarding nicotine's critical defensive role in nature and highlights the tight link between nicotine biosynthesis and gene expression levels of quantitative resistance-related genes for better environmental adaptation.

Determination of tobacco-specific nitrosamines in tobacco and mainstream cigarette smoke using one-step clean-up coupled with liquid chromatography- tandem mass spectrometry.

A method for determining tobacco-specific nitrosamines (TSNAs) in tobacco and cigarette smoke using liquid chromatography-tandem mass spectrometry was established. The established method amended the deficiencies that exist in current mainstream methods. In this method, TSNAs in tobacco and cigarette smoke were extracted by water. The aqueous extract was then extracted by dichloromethane, and the extract could be analyzed by liquid chromatography-tandem mass spectrometry after a solvent replacement. This method was used to analyze flue-cured tobacco samples, and the response of the target compounds was about 10 times higher than that of the ammonium acetate extraction method. When analyzing cigarette smoke samples, the response strength and chromatographic peak purity of the target compounds were also significantly improved. The proposed method exhibited good linearities for both tobacco and cigarette smoke samples (r2 > 0.99). The limits of detection (LODs) for tobacco and cigarette smoke samples were 0.2-1.0 ng/g and 0.1-0.3 ng/cigarette, respectively. Additionally, this method exhibited desirable accuracy and precision. The TSNAs recovery values from tobacco and cigarette smoke samples ranged from 95.7 % to 107.7 % with inter- and intra-day relative standard deviations (RSDs) of less than 7.4 %. This method is simple, effective, and has wide adaptability. It is a useful upgrade to the existing methods for analyzing TSNAs in tobacco and cigarette smoke.

Ethylene inhibits ABA-induced stomatal closure via regulating NtMYB184-mediated flavonol biosynthesis in tobacco.

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.

Phytomelatonin interferes with flavonols biosynthesis to regulate ROS production and stomatal closure in tobacco.

Flavonols are well-known antioxidants that prevent stomatal closure via interfering with ROS signaling. Phytomelatonin regulates stomatal closure, but the signaling pathways are still largely unknown. Here, we investigated the role of flavonols in phytomelatonin-mediated stomatal closure in tobacco plants. The application of melatonin induced stomatal closure through NADPH oxidase-mediated ROS production. Transgenic tobacco plants overexpressing soybean GmSNAT1 (coding for serotonin N-acetyltransferase that catalyzes the penultimate step in phytomelatonin biosynthesis) had higher phytomelatonin concentration, accumulated more ROS in guard cells and were more sensitive to melatonin-induced stomatal closure than the wild-type plants, which was associated with the higher expression of PMTR1-homologous genes. Exogenous melatonin decreased flavonol concentrations in guard cells and the expression of flavonoid-related genes in wild-type and transgenic tobacco plants, and these inhibitory effects were more obvious in GmSNAT1-overexpressing plants than the wild type. However, the melatonin-mediated stomatal closure and ROS production were diminished by the application of kaempferol (a type of flavonol). Additionally, transgenic tobacco plants with increased expression of NtFLS (encoding flavonol synthase) were less sensitive to melatonin-induced stomatal closure. In conclusion, phytomelatonin hampers the biosynthesis of flavonols in guard cells, which results in high concentration of ROS and induces stomatal closure in tobacco plants.

Integrative analysis of sensory evaluation and non-targeted metabolomics to unravel tobacco leaf metabolites associated with sensory quality of heated tobacco.

Introduction: Heated tobacco (Nicotiana tabacum L.) products are heating tobacco plug at a temperature of 350°C and produce different emissions in aerosol and sensory perceptions of tobacco leaf compared with combustible tobacco. Previous study assessed different tobacco varieties in heated tobacco for sensory quality and analyzed the links between sensory scores of the final products and certain chemical classes in tobacco leaf. However, contribution of individual metabolites to sensory quality of heated tobacco remains largely open for investigation. Methods: In present study, five tobacco varieties were evaluated as heated tobacco for sensory quality by an expert panel and the volatile and non-volatile metabolites were analyzed by non-targeted metabolomics profiling. Results: The five tobacco varieties had distinct sensory qualities and can be classified into higher and lower sensory rating classes. Principle component analysis and hierarchical cluster analysis showed that leaf volatile and non-volatile metabolome annotated were grouped and clustered by sensory ratings of heated tobacco. Orthogonal projections to latent structures discriminant analysis followed by variable importance in projection and fold-change analysis revealed 13 volatiles and 345 non-volatiles able to discriminate the tobacco varieties with higher and lower sensory ratings. Some compounds such as ß-damascenone, scopoletin, chlorogenic acids, neochlorogenic acids, and flavonol glycosyl derivatives had strong contribution to the prediction of sensory quality of heated tobacco. Several lyso-phosphatidylcholine and lyso-phosphatidylethanolamine lipid species, and reducing and non-reducing sugar molecules were also positively related to sensory quality. Discussion: Taken together, these discriminating volatile and non-volatile metabolites support the role of leaf metabolites in affecting the sensory quality of heated tobacco and provide new information on the types of leaf metabolites that can be used to predict applicability of tobacco varieties for heated tobacco products.

Systematic Identification of Methyl Jasmonate-Responsive Long Noncoding RNAs and Their Nearby Coding Genes Unveils Their Potential Defence Roles in Tobacco BY-2 Cells.

Long noncoding RNAs (lncRNAs) are distributed in various species and play critical roles in plant growth, development, and defence against stimuli. However, the lncRNA response to methyl jasmonate (MeJA) treatment has not been well characterized in Nicotiana tabacum Bright Yellow-2 (BY-2) cells, and their roles in plant defence remain elusive. Here, 7848 reliably expressed lncRNAs were identified in BY-2 cells, of which 629 differentially expressed (DE) lncRNAs were characterized as MeJA-responsive lncRNAs. The lncRNAs in BY-2 cells had a strong genus specificity in Nicotiana. The combined analysis of the cis-regulated lncRNAs and their target genes revealed the potential up- and downregulated target genes that are responsible for different biological functions and metabolic patterns. In addition, some lncRNAs for response-associated target genes might be involved in plant defence and stress resistance via their MeJA- and defence-related cis-regulatory elements. Moreover, some MeJA-responsive lncRNA target genes were related to quinolinate phosphoribosyltransferase, lipoxygenases, and endopeptidase inhibitors, which may contribute to nicotine synthesis and disease and insect resistance, indicating that MeJA-responsive lncRNAs regulate nicotine biosynthesis and disease resistance by regulating their potential target genes in BY-2 cells. Therefore, our results provide more targets for genetically engineering the nicotine content and plant defence in tobacco plants.

Physiological and Molecular Changes in Cherry Red Tobacco in Response to Iron Deficiency Stress.

The genotype CR60 is a spontaneous Cherry Red variant (containing granular red dapples on flue-cured leaves) of the Yunyan 87 (Y87) tobacco; it accumulates higher concentration of iron (Fe) in leaves than Y87, but the physiological differences between them remain largely unknown. We investigated the physiological and molecular mechanisms of CR60 in response to Fe deficiency under hydroponic conditions. Our results showed no significant phenotypic difference between Y87 and CR60 at optimal (40 µM) and high Fe (160 and 320 µM) concentrations. By contrast, CR60 exhibited higher tolerance to Fe deficiency (0 µM) than Y87, as shown by higher concentrations of chlorophyll in CR60 leaves after 21-day Fe-deficiency stress. Transcriptome profiling coupled with RT-PCR analyses found that the expression of IRT1 and several genes associated with chlorophyll biosynthesis and photosynthesis (e.g., PRO, GSA, FD1, PsbO, and PC) was higher in CR60 than Y87. These results indicated that CR60 maintains sufficient Fe uptake, chlorophyll biosynthesis and photosynthetic rate when subjected to Fe starvation.

A spatial-temporal understanding of gene regulatory networks and NtARF-mediated regulation of potassium accumulation in tobacco.

MAIN CONCLUSION: After tobacco topping, changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the NtARF genes, thus causing changes in K+ content. Tobacco (Nicotiana tabacum) is a valuable industrial and commercial crop, and the leaf is its primary product. Topping (removing apical buds) is a common agronomic practice that significantly improves the yield of tobacco leaves. Potassium (K+) plays an important physiological role in tobacco growth and leaf traits, including combustibility, aroma, and safety in cigarette products, and its levels are significantly decreased after topping. Here, to present global spatial-temporal gene expression profiles and gene regulatory networks of the core elements of K+ uptake, leaves and roots from topped and untopped plants at short- and long-term time points after topping were sampled for transcriptome analysis. We found that the wounding response was initiated in leaves in the early stages after topping. Then, in the long term, processes related to metabolism and transcription regulation, as well as ion binding and transport, were altered. The expression profiles showed that core elements of K+ uptake and xylem loading were drastically suppressed in roots after topping. Finally, transient expression experiments confirmed that changes in the auxin content could affect K+ uptake by inhibiting the activity of K+ uptake-related genes through the tobacco auxin response factor (NtARF) genes, thus causing changes in the K+ content. These results suggest that some ARFs could be selected as targets to enhance the expressions of K+ uptake transporters, leading to increment of K+ contents and improvement of leaf quality in tobacco breeding.

Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.).

KEY MESSAGE: NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco.

NtAIDP1, a novel NtJAZ interacting protein, binds to an AT-rich region to activate the transcription of jasmonate-inducible genes in tobacco.

In plants, jasmonate ZIM-domain proteins (JAZs) act as critical regulators, interacting physically with transcription factors (TFs) and other transcriptional regulators to modulate jasmonate (JA)-responsive gene expression and participate in crosstalk with other hormone signalling pathways. Identifying novel JAZ-interacting proteins will provide new insights into JA signalling cascades in plants. Here, we performed yeast two-hybrid screening to identify 70 NtJAZ1-interacting proteins, including an A/T-rich interaction domain containing protein 1 (NtAIDP1) from JA-treated tobacco Bright Yellow-2 (BY-2) cells. NtAIDP1 is localised in the nucleus and interacts with NtJAZ1 via its C-terminal heat shock protein 20 (HSP) domain. Aside from NtJAZ1, NtAIDP1 also interacts with other JA-inducible NtJAZs, including NtJAZ2b, NtJAZ2b.2, NtJAZ5, NtJAZ7, NtJAZ11 and NtJAZ12, but not with NtJAZ3, NtJAZ3b or NtJAZ10, and interacts with NtNINJA, NtDELLA1 and NtDELLA2 in the yeast two-hybrid assay. Furthermore, NtAIDP1 binds to the AT-rich region of the GAG fragment of the putrescine N-methyltransferase 1a (NtPMT1a) promoter and activates the transcriptional activity of the GAG fragment, whereas NtMYC2a interacts with and competitively inhibits the transactivational activity of NtAIDP1 in Arabidopsis mesophyll protoplasts. Overexpression of NtAIDP1 promotes the transcription of NtPDF1.2 and NtJAZ1, but has little effect on the expression of NtPMT1a, quinolinate phosphoribosyltransferase 2 (NtQPT2), and NtMYC2a in tobacco. These results indicate that NtAIDP1 is a new component of the JA signalling pathway and is involved in JA-regulated gene expression.

R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.).

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

Metabolome and transcriptome analyses of the molecular mechanisms of flower color mutation in tobacco.

BACKGROUND: Anthocyanins determinate the flower color of many plants. Tobacco is a model plant for studying the molecular regulation of flower coloration. We investigated the mechanism underlying flower coloration in tobacco by profiling flavonoid metabolites,expression of anthocyanin biosynthetic structural genes and their regulator genes in the pink-flowered tobacco cultivar Yunyan 87 and white-flowered Yunyan 87 mutant. RESULT: Significant down-accumulation of anthocyanins, including cyanidin 3-O-glucoside, cyanin, cyanidin 3-O-rutinoside, pelargonidin 3-O-beta-D-glucoside, cyanidin O-syringic acid, pelargonin, and pelargonidin 3-O-malonylhexoside (log2 fold change < - 10), endowed the flower color mutation in Yunyan 87 mutant. Transcriptome analysis showed that the coordinately down-regulated anthocyanin biosynthetic genes including chalcone isomerase, naringenin 3-dioxygenase, dihydroflavonol 4-reductase and UDP-glucose:flavonoid 3-O-glucosyltransferase played critical roles in suppressing the formation of the aforesaid anthocyanins. Several genes encoding MYB and bHLH transcription factors were also found down-regulated, and probably the reason for the suppression of structural genes. CONCLUSION: This is the first study of tobacco flower coloration combining metabolome and transcriptome analyses, and the results shed a light on the systematic regulation mechanisms of flower coloration in tobacco. The obtained information will aid in developing strategies to modify flower color through genetic transformation.

Daily rhythms of phytomelatonin signaling modulate diurnal stomatal closure via regulating reactive oxygen species dynamics in Arabidopsis.

Melatonin is a well-studied neurohormone oscillating in a 24-h cycle in vertebrates. Phytomelatonin is widespread in plant kingdom, but it remains elusive whether this newly characterized putative hormone underlies the regulation by daily rhythms. Here, we report phytomelatonin signaling, as reflected by changes in endogenous concentrations of phytomelatonin and expression of genes associated with biosynthesis of phytomelatonin (AtSNAT1, AtCOMT1, and AtASMT) and its receptor (AtPMTR1), shows 24-h oscillations in Arabidopsis. The variation of reactive oxygen species (ROS) production and scavenging and expression of ROS-related genes significantly decrease in pmtr1 and snat and increase in PMTR1-OE seedlings, indicating the rhythmicity in phytomelatonin signaling is required for maintenance of ROS dynamics. Additionally, the ROS signaling feedback influences the expression of AtSNAT1, AtCOMT1, AtASMT, and AtPMTR1, suggesting the phytomelatonin and ROS signaling are coordinately interrelated. The pmtr1 mutant plants lose diurnal stomatal closure, with stomata remaining open during daytime as well as nighttime and mutants showing more water loss and drought sensitivity when compared with the wild-type Col-0 plants. Taken together, our results suggest that PMTR1-regulated ROS signaling peaks in the afternoon and may transmit the darkness signals to trigger stomatal closure, which might be essential for high water-use efficiency and drought tolerance.

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

An efficient TILLING platform for cultivated tobacco.

Targeting-induced local lesions in genomes (TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants. Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco (Nicotiana tabacum L.) using an ethyl methanesulfonate (EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation, leaf tissue sampling, DNA extraction, normalization, pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106 kb on average. Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform (available at http://www.croptilling.org) can be used in tobacco to facilitate functional genomics applications.

Ethylene response factor NtERF91 positively regulates alkaloid accumulations in tobacco (Nicotiana tabacum L.).

Tobacco alkaloid metabolism is regulated by various transcription factors (TFs). Here, we have characterized a non-NIC2 locus gene, Ethylene Response Factor 91 (ERF91), function in regulation of alkaloid accumulation in tobacco. NtERF91 was preferentially expressed in roots and induced by jasmonic acid. Additionally, NtERF91 was able to in vitro bind to the NtPMT2 and NtQPT2 promoters via directly targeting the GCC-box elements and transactivate NtQPT2 gene expression. Ectopic overexpression of NtERF91 not only increased the expression of most nicotine biosynthetic genes, but also altered alkaloid accumulation profile, resulting in dramatically anatabine accumulation. We conclude that NtERF91 plays an overlapped but distinct role in regulating tobacco alkaloid accumulations.

Genome-wide analysis of the HAK potassium transporter gene family reveals asymmetrical evolution in tobacco (Nicotiana tabacum).

Being an essential mineral nutrient, potassium (K+) plays numerous important roles in plant growth and development and determines the yield and quality of crop products. The cellular level of K+ is controlled to a large extent by the K+ transporter, which belongs to the KT/HAK/KUP (HAK) family. However, little is known about these genes in tobacco. In this study, we surveyed the tobacco genome and identified 41 putative NtHAK genes (NtHAKS1-NtHAKS21 and NtHAKT1-NtHAKT20). Investigation of the cis-elements in upstream regions of these NtHAK genes suggests that members of this family respond to environmental cues and phytohormones. Expression data mining reveals that NtHAK genes showed clear sub-genome dominance. In all, these results will provide molecular insights into K+ transporter research in tobacco.

Characterization of Nicotiana tabacum genotypes possessing deletion mutations that affect potyvirus resistance and the production of trichome exudates.

BACKGROUND: Advances in genomics technologies are making it increasingly feasible to characterize breeding lines that carry traits of agronomic interest. Tobacco germplasm lines that carry loci designated VAM and va have been extensively investigated due to their association with potyvirus resistance (both VAM and va) and defects in leaf surface compounds originating from glandular trichomes (VAM only). Molecular studies and classical genetic analyses are consistent with the model that VAM and va represent deletion mutations in the same chromosomal region. In this study, we used RNA-seq analysis, together with emerging tobacco reference genome sequence information to characterize the genomic regions deleted in tobacco lines containing VAM and va. RESULTS: Tobacco genotypes TI 1406 (VAM), K326-va and K326 (wild type) were analyzed using RNA-seq to generate a list of genes differentially expressed in TI 1406 and K326-va, versus the K326 control. Candidate genes were localized onto tobacco genome scaffolds and validated as being absent in only VAM, or missing in both VAM and va, through PCR analysis. These results enabled the construction of a map that predicted the relative extent of the VAM and va mutations on the distal end of chromosome 21. The RNA-seq analyses lead to the discovery that members of the cembratrienol synthase gene family are deleted in TI 1406. Transformation of TI 1406 with a cembratrienol synthase cDNA, however, did not recover the leaf chemistry phenotype. Common to both TI 1406 and K326-va was the absence of a gene encoding a specific isoform of a eukaryotic translation initiation factor (eiF4E1.S). Transformation experiments showed that ectopic expression of eiF4E1.S is sufficient to restore potyvirus susceptibility in plants possessing either the va or VAM mutant loci. CONCLUSIONS: We have demonstrated the feasibility of using RNA-seq and emerging whole genome sequence resources in tobacco to characterize the VAM and va deletion mutants. These results lead to the discovery of genes underlying some of the phenotypic traits associated with these historically important loci. Additionally, initial size estimations were made for the deleted regions, and dominant markers were developed that are very close to one of the deletion junctions that defines va.