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 (log₂fold 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.

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.

Stomatal movement could be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers biosynthesis of antioxidant flavonol to suppress ROS accumulation and prevent ABA-induced stomatal closure, but the underlying mechanism remains largely unknown. We isolated and characterized tobacco R2R3-MYB NtMYB184, belonging to the flavonol-specific SG7 subgroup. The RNAi suppression (KD line) and knockout (myb184) of NtMYB184 caused a down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonol in tobacco leaves. Yeast one-hybrid assay, transactivation assay, EMSA and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via the MYBPLANT motifs. In tobacco, NtMYB184 regulates 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 the 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 guard cells.

Rutin, one of the metabolites of the flavonoid pathway, shows great potential in industrial applications as a key component in pharmaceutical medicines and biological pesticides. Although the genetic manipulation of transcription factors (TFs) could increase rutin levels in plants, the accompanying accumulation of structurally similar chemicals complicates industrial rutin extraction. In this study, we demonstrated remarkably elevated rutin content (3.5-4.4-fold relative to controls) in transgenic tobacco plants by overexpressing NtFLS2. The levels of other intermediates in the branch pathway did not change much except for a moderate increase of kaempferol-3-O-rutinoside. Furthermore, the transcript levels of pathway genes in transgenic lines were comparable with controls, indicating genetic engineering did not significantly alter the branch pathway. Additionally, the transgenic tobacco plants appeared normal except for a flower color change from light red to white suggesting that it could be a valuable material for industrial extraction of rutin.

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. 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. 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.