The Genetic Basis of Flower Color Differences in Nicotiana tabacum

  • Elizabeth W. McCarthy
  • Jacob B. Landis
  • Amelda Kurti
  • Amber J. Lawhorn
  • Amy LittEmail author
Part of the Compendium of Plant Genomes book series (CPG)


Nicotiana tabacum L accessions vary in flower color from light pink to magenta. The differences in flower color are attributable to differences in anthocyanin content. To determine the genetic basis of flower color differences, we generated transcriptomes and quantified transcript levels of flavonoid biosynthetic genes in four N. tabacum accessions and their diploid progenitors. High expression ratios of the flavonol synthase (FLS) gene to dihydroflavonol 4-reductase (DFR) gene are found in light-pink flowers, suggesting that competition between the FLS and DFR enzymes for the same substrates may drive the flux of the flavonoid biosynthetic pathway toward producing flavonols at the expense of anthocyanins, resulting in light-pink flowers. The high FLS:DFR expression ratio appears to be attributable to DFR activation later in development in light-pink flowers.


Anthocyanin Flavonol Flower color Nicotiana Tobacco 



We thank James Giovannoni and Yimin Xu for transcriptome sequencing, Zhangjun Fei for help with sequence analysis, Loyal Goff for assistance with cummerbund, and Christopher Fiscus, Dinusha Maheepala, Glen Morrison, and Alex Rajewski for help with R programming.


  1. Bai Y, Pattanaik S, Patra B et al (2011) Flavonoid-related basic helix-loop-helix regulators, NtAn1a and NtAn1b, of tobacco have originated from two ancestors and are functionally active. Planta 234:363–375CrossRefGoogle Scholar
  2. Cao L, Rohart F, Gonzalez I et al (2016) mixOmics: Omics data integration project. R package version 6(1):1Google Scholar
  3. Chase MW, Knapp S, Cox AV et al (2003) Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae). Ann Bot 92:107–127CrossRefGoogle Scholar
  4. Chen H, Boutros PC (2011) VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinform 12:35CrossRefGoogle Scholar
  5. Clarkson JJ, Dodsworth S, Chase MW (2017) Time-calibrated phylogenetic trees establish a lag between polyploidisation and diversification in Nicotiana (Solanaceae). Plant Syst Evol 303:1001–1012CrossRefGoogle Scholar
  6. Clarkson JJ, Kelly LJ, Leitch AR et al (2010) Nuclear glutamine synthetase evolution in Nicotiana: phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids. Mol Phylogenet Evol 55:99–112CrossRefGoogle Scholar
  7. Clarkson JJ, Knapp S, Garcia VF et al (2004) Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions. Mol Phylogenet Evol 33:75–90CrossRefGoogle Scholar
  8. Crosby KC, Pietraszewska-Bogiel A, Gadella TWJ Jr, Winkel BSJ (2011) Förster resonance energy transfer demonstrates a flavonoid metabolon in living plant cells that displays competitive interactions between enzymes. FEBS Lett 585:2193–2198CrossRefGoogle Scholar
  9. Davies KM, Schwinn KE, Deroles SC et al (2003) Enhancing anthocyanin production by altering competition for substrate between flavonol synthase and dihydroflavonol 4-reductase. Euphytica 131:259–268CrossRefGoogle Scholar
  10. Des Marais DL, Rausher MD (2010) Parallel evolution at multiple levels in the origin of hummingbird pollinated flowers in Ipomoea. Evolution 64:2044–2054PubMedGoogle Scholar
  11. Edwards KD, Fernandez-Pozo N, Drake-Stowe K et al (2017) A reference genome for Nicotiana tabacum enables map-based cloning of homeologous loci implicated in nitrogen utilization efficiency. BMC Genom 18:448CrossRefGoogle Scholar
  12. Finn RD, Coggill P, Eberhardt RY et al (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–85CrossRefGoogle Scholar
  13. Gates DJ, Olson BJSC, Clemente TE, Smith SD (2017) A novel R3 MYB transcriptional repressor associated with the loss of floral pigmentation in Iochroma. New Phytol. Scholar
  14. Goff L, Trapnell C, Kelley D (2013) cummeRbund: analysis, exploration, manipulation, and visualization of Cufflinks high-throughput sequencing data. R package versionGoogle Scholar
  15. Goodspeed TH (1954) The genus Nicotiana. Chronica Botanica, Waltham, Massachusetts, USAGoogle Scholar
  16. Grabherr MG, Haas BJ, Yassour M et al (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652CrossRefGoogle Scholar
  17. Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780CrossRefGoogle Scholar
  18. Haas BJ, Papanicolaou A, Yassour M et al (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512CrossRefGoogle Scholar
  19. Hoballah ME, Gübitz T, Stuurman J et al (2007) Single gene-mediated shift in pollinator attraction in Petunia. Plant Cell 19:779–790CrossRefGoogle Scholar
  20. Holton T, Brugliera F, Tanaka Y (1993) Cloning and expression of flavonol synthase in Petunia hybrida. Plant J 4:1003–1010CrossRefGoogle Scholar
  21. Joshi NA, Fass JN (2011) Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33) [Software]Google Scholar
  22. Knapp S, Chase MW, Clarkson JJ (2004) Nomenclatural changes and a new sectional classification in Nicotiana (Solanaceae). Taxon 53:73–82CrossRefGoogle Scholar
  23. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359CrossRefGoogle Scholar
  24. Luo P, Ning G, Wang Z et al (2015) Disequilibrium of flavonol synthase and dihydroflavonol-4-reductase expression associated tightly to white vs. red color flower formation in plants. Front Plant Sci 6:1257Google Scholar
  25. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17:10–12Google Scholar
  26. McCarthy EW, Arnold SEJ, Chittka L et al (2015) The effect of polyploidy and hybridization on the evolution of floral colour in Nicotiana (Solanaceae). Ann Bot 115:1117–1131CrossRefGoogle Scholar
  27. McCarthy EW, Berardi AE, Smith SD, Litt A (2017) Related allopolyploids display distinct floral pigment profiles and transgressive pigments. Am J Bot 104:92–101CrossRefGoogle Scholar
  28. Pattanaik S, Kong Q, Zaitlin D et al (2010) Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco. Planta 231:1061–1076CrossRefGoogle Scholar
  29. Quattrocchio F, Wing J, van der Woude K et al (1999) Molecular analysis of the anthocyanin2 gene of Petunia and its role in the evolution of flower color. Plant Cell 11:1433–1444CrossRefGoogle Scholar
  30. Rasband WS (1997) ImageJ. US National Institutes of Health, Bethesda, MDGoogle Scholar
  31. Ritchie ME, Phipson B, Wu D et al (2015) limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43:e47CrossRefGoogle Scholar
  32. Schwinn K, Venail J, Shang Y et al (2006) A small family of MYB-regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18:831–851CrossRefGoogle Scholar
  33. Sheehan H, Moser M, Klahre U et al (2016) MYB-FL controls gain and loss of floral UV absorbance, a key trait affecting pollinator preference and reproductive isolation. Nat Genet 48:159–166CrossRefGoogle Scholar
  34. Sierro N, Battey JND, Ouadi S et al (2013) Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis. Genome Biol 14:R60CrossRefGoogle Scholar
  35. Smith SD, Rausher MD (2011) Gene loss and parallel evolution contribute to species difference in flower color. Mol Biol Evol 28:2799–2810CrossRefGoogle Scholar
  36. Stracke R, De Vos RCH, Bartelniewoehner L et al (2009) Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta 229:427–445CrossRefGoogle Scholar
  37. Streisfeld MA, Rausher MD (2009) Genetic changes contributing to the parallel evolution of red floral pigmentation among Ipomoea species. New Phytol 183:751–763CrossRefGoogle Scholar
  38. The UniProt Consortium (2017) UniProt: the universal protein knowledgebase. Nucleic Acids Res 45:D158–D169CrossRefGoogle Scholar
  39. Trabelsi N, Petit P, Manigand C et al (2008) Structural evidence for the inhibition of grape dihydroflavonol 4-reductase by flavonols. Acta Crystallogr D Biol Crystallogr D64:883–891CrossRefGoogle Scholar
  40. Trapnell C, Roberts A, Goff L et al (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578CrossRefGoogle Scholar
  41. Warnes GR, Bolker B, Bonebakker L, Gentleman R (2009) gplots: Various R programming tools for plotting data. R package versionGoogle Scholar
  42. Wessinger CA, Rausher MD (2015) Ecological transition predictably associated with gene degeneration. Mol Biol Evol 32:347–354CrossRefGoogle Scholar
  43. Wickham H (2009) ggplot2: Elegant Graphics for Data Analysis Springer-Verlag. New YorkGoogle Scholar
  44. Yuan Y-W, Rebocho AB, Sagawa JM et al (2016) Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species. Proc Natl Acad Sci U S A 113:2448–2453CrossRefGoogle Scholar
  45. Yuan Y-W, Sagawa JM, Young RC et al (2013) Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus. Genetics 194:255–263CrossRefGoogle Scholar
  46. Zhong S, Joung J-G, Zheng Y et al (2011) High-throughput illumina strand-specific RNA sequencing library preparation. Cold Spring Harb Protoc 2011:940–949CrossRefGoogle Scholar
  47. Zufall RA, Rausher MD (2004) Genetic changes associated with floral adaptation restrict future evolutionary potential. Nature 428:847–850CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  • Elizabeth W. McCarthy
    • 1
    • 2
  • Jacob B. Landis
    • 1
    • 3
  • Amelda Kurti
    • 1
  • Amber J. Lawhorn
    • 1
  • Amy Litt
    • 1
    Email author
  1. 1.Department of Botany and Plant SciencesUniversity of CaliforniaRiverside, RiversideUSA
  2. 2.Department of Biological SciencesSUNY CortlandCortlandUSA
  3. 3.School of Integrative Plant Science, Section of Plant Biology and the L.H. Bailey HortoriumCornell UniversityIthacaUSA

Personalised recommendations