, Volume 224, Issue 4, pp 750–760 | Cite as

Evolution of proteinase inhibitor defenses in North American allopolyploid species of Nicotiana

  • Jianqiang Wu
  • Christian Hettenhausen
  • Ian T. Baldwin
Original Article


We studied the jasmonate (JA)-elicited trypsin-proteinase inhibitor (TPI) anti-herbivore defense system in North American Nicotiana to understand how complex polygenetic traits evolve after allopolyploidy speciation. N. quadrivalvis (Nq) and N. clevelandii (Nc) are allotetraploid descendant species of the ancestors of the diploid species N. attenuata (Na) and N. obtusifolia (No). From cDNA, intron and promoter sequence analyses, and Southern blotting, we deduced that only the maternally derived No TPI genes were retained in the tetraploid genomes (Nq, Nc), whereas the sequences of the paternal Na ancestor were deleted. The number of TPI repeats in different Nicotiana taxa was independent of phylogenetic associations. In Na, TPI activity and mRNA transcript accumulation as well as JA levels increased dramatically above wound-induced levels when the oral secretions (OS) from Manduca sexta larvae were introduced into wounds. This OS-mediated amplification of defense signaling and downstream response was also found in the tetraploid genomes but was absent from No; in No, OS treatment suppresses TPI mRNA accumulation and activity and does not increase JA accumulation. Hence, the tetraploids retained components of Na’s signaling system, but lost Na’s TPI genes and used No’s TPI genes to retain a functional TPI defense system, underscoring the genomic flexibility that enables complex polygenic traits to be retained in allopolyploid species.


Nicotiana Plant–herbivore interaction Polygenic adaptation Polyploidy Proteinase inhibitor Solanaceae 



Analysis of variance


High-performance liquid chromatography coupled tandem mass spectrometry


Internal transcribed spacer




Nicotiana attenuata


N. clevelandii


N. obtusifolia


N. quadrivalvis


Oral secretions


Trypsin-proteinase inhibitor



We thank Max-Planck Gesellschaft and the Deutsche Forschungsgemeinschaft (SPP1152) for providing funding for this work.

Supplementary material

425_2006_256_MOESM1_ESM.pdf (196 kb)
Supplementary material


  1. Adams KL, Wendel JF (2005) Polyploidy and genome evolution in plants. Curr Opin Plant Biol 8:135–141PubMedCrossRefGoogle Scholar
  2. Atkinson AH, Heath RL, Simpson RJ, Clarke AE, Anderson MA (1993) Proteinase inhibitors in Nicotiana alata stigmas are derived from a precursor protein which is processed into five homologous inhibitors. Plant Cell 5:203–213PubMedCrossRefGoogle Scholar
  3. Barta E, Pintar A, Pongor S (2002) Repeats with variations: accelerated evolution of the Pin2 family of proteinase inhibitors. Trends Genet 18:600–603PubMedCrossRefGoogle Scholar
  4. Blanc G, Wolfe KH (2004) Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolution. Plant Cell 16:1679–1691PubMedCrossRefGoogle Scholar
  5. Chase MW, Knapp S, Cox AV, Clarkson JJ, Butsko Y, Joseph J, Savolainen V, Parokonny AS (2003) Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae). Ann Bot 92:107–127PubMedCrossRefGoogle Scholar
  6. Choi D, Park JA, Seo YS, Chun YJ, Kim WT (2000) Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L. Biochim Biophys Acta 1492:211–215PubMedGoogle Scholar
  7. Clarkson JJ, Knapp S, Garcia VF, Olmstead RG, Leitch AR, Chase MW (2004) Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions. Mol Phylogenet Evol 33:75–90PubMedCrossRefGoogle Scholar
  8. van Dam NM, Horn M, Mares M, Baldwin IT (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. J Chem Ecol 27:547–568PubMedCrossRefGoogle Scholar
  9. Doebley J, Lukens L (1998) Transcriptional regulators and the evolution of plant form. Plant Cell 10:1075–1082PubMedCrossRefGoogle Scholar
  10. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  11. Feldman M, Liu B, Segal G, Abbo S, Levy AA, Vega JM (1997) Rapid elimination of low-copy DNA sequences in polyploid wheat: a possible mechanism for differentiation of homoeologous chromosomes. Genetics 147:1381–1387PubMedGoogle Scholar
  12. Glawe GA, Zavala JA, Kessler A, van Dam NM, Baldwin IT (2003) Ecological costs and benefits correlated with trypsin protease inhibitor production in Nicotiana attenuata. Ecology 84:79–90CrossRefGoogle Scholar
  13. Goodspeed TH (1954) The genus Nicotiana. Chronica Botanica, WalthamGoogle Scholar
  14. Halitschke R, Baldwin IT (2003) Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata. Plant J 36:794–807PubMedCrossRefGoogle Scholar
  15. Halitschke R, Schittko U, Pohnert G, Boland W, Baldwin IT (2001) Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid–amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses. Plant Physiol 125:711–717PubMedCrossRefGoogle Scholar
  16. Han FP, Fedak G, Ouellet T, Liu B (2003) Rapid genomic changes in interspecific and intergeneric hybrids and allopolyploids of Triticeae. Genome 46:716–723PubMedCrossRefGoogle Scholar
  17. Heath RL, Barton PA, Simpson RJ, Reid GE, Lim G, Anderson MA (1995) Characterization of the protease processing sites in a multidomain proteinase inhibitor precursor from Nicotiana alata. Eur J Biochem 230:250–257PubMedCrossRefGoogle Scholar
  18. Heidel AJ, Baldwin IT (2004) Microarray analysis of salicylic acid- and jasmonic acid-signalling in responses of Nicotiana attenuata to attack by insects from multiple feeding guilds. Plant Cell Environ 27:1362–1373CrossRefGoogle Scholar
  19. Helentjaris T, Weber D, Wright S (1988) Identification of the genomic locations of duplicate nucleotide-sequences in maize by analysis of restriction fragment length polymorphisms. Genetics 118:353–363PubMedGoogle Scholar
  20. Horn M, Patankar AG, Zavala JA, Wu J, Doleckova-Maresova L, Vujtechova M, Mares M, Baldwin IT (2005) Differential elicitation of two processing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata. Plant Physiol 139:375–388PubMedCrossRefGoogle Scholar
  21. Kashkush K, Feldman M, Levy AA (2002) Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics 160:1651–1659PubMedGoogle Scholar
  22. Kellis M, Birren BW, Lander ES (2004) Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428:617–624PubMedCrossRefGoogle Scholar
  23. Kim CY, Liu Y, Thorne ET, Yang H, Fukushige H, Gassmann W, Hildebrand D, Sharp RE, Zhang S (2003) Activation of a stress-responsive mitogen-activated protein kinase cascade induces the biosynthesis of ethylene in plants. Plant Cell 15:2707–2718PubMedCrossRefGoogle Scholar
  24. Koiwa H, Bressan RA, Hasegawa PM (1997) Regulation of protease inhibitors and plant defense. Trends Plant Sci 2:379–384CrossRefGoogle Scholar
  25. Krügel T, Lim M, Gase K, Halitschke R, Baldwin IT (2002) Agrobacterium-mediated transformation of Nicotiana attenuata, a model ecological expression system. Chemoecology 12:177–183CrossRefGoogle Scholar
  26. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163PubMedCrossRefGoogle Scholar
  27. Lai J, Ma J, Swigonova Z, Ramakrishna W, Linton E, Llaca V, Tanyolac B, Park YJ, Jeong OY, Bennetzen JL, Messing J (2004) Gene loss and movement in the maize genome. Genome Res 14:1924–1931PubMedCrossRefGoogle Scholar
  28. Langkjaer RB, Cliften PF, Johnston M, Piskur J (2003) Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. Nature 421:848–852PubMedCrossRefGoogle Scholar
  29. Lee MC, Scanlon MJ, Craik DJ, Anderson MA (1999) A novel two-chain proteinase inhibitor generated by circularization of a multidomain precursor protein. Nat Struct Biol 6:526–530PubMedCrossRefGoogle Scholar
  30. Leitch IJ, Bennett MD (1997) Polyploidy in angiosperms. Trends Plant Sci 2:470–476CrossRefGoogle Scholar
  31. Levin DA (1983) Polyploidy and novelty in flowering plants. Am Nat 122:1–25CrossRefGoogle Scholar
  32. Liu B, Vega JM, Feldman M (1998a) Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. II. Changes in low-copy coding DNA sequences. Genome 41:535–542CrossRefGoogle Scholar
  33. Liu B, Vega JM, Segal G, Abbo S, Rodova H, Feldman M (1998b) Rapid genomic changes in newly synthesized amphiploids of Triticum and Aegilops. I. Changes in low-copy noncoding DNA sequences. Genome 41:272–277CrossRefGoogle Scholar
  34. Lou Y, Baldwin IT (2003) Manduca sexta recognition and resistance among allopolyploid Nicotiana host plants. Proc Natl Acad Sci USA 100(Suppl 2):14581–14586PubMedCrossRefGoogle Scholar
  35. Madlung A, Tyagi AP, Watson B, Jiang H, Kagochi T, Doerge RW, Martienssen R, Comai L (2005) Genomic changes in synthetic Arabidopsis polyploids. Plant J 41:221–230PubMedCrossRefGoogle Scholar
  36. Masterson J (1994) Stomatal size in fossil plants: evidence for polyploidy in the majority of angiosperms. Science 264:421–424PubMedCrossRefGoogle Scholar
  37. Miller EA, Lee MC, Atkinson AH, Anderson MA (2000) Identification of a novel four-domain member of the proteinase inhibitor II family from the stigmas of Nicotiana alata. Plant Mol Biol 42:329–333PubMedCrossRefGoogle Scholar
  38. Murren CJ, Pigliucci M (2005) Morphological responses to simulated wind in the genus Brassica (Brassicaceae): allopolyploids and their parental species. Am J Bot 92:810–818CrossRefGoogle Scholar
  39. O’Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ (1996) Ethylene as a signal mediating the wound response of tomato plants. Science 274:1914–1917PubMedCrossRefGoogle Scholar
  40. Ohno S (1970) Evolution by gene duplication. Springer, Berlin Heidelberg New YorkGoogle Scholar
  41. Otto SP, Whitton J (2000) Polyploid incidence and evolution. Annu Rev Genet 34:401–437PubMedCrossRefGoogle Scholar
  42. Pearce G, Johnson S, Ryan CA (1993) Purification and characterization from tobacco (Nicotiana tabacum) leaves of six small, wound-inducible, proteinase isoinhibitors of the potato inhibitor II family. Plant Physiol 102:639–644PubMedCrossRefGoogle Scholar
  43. Pena-Cortes H, Willmitzer L, Sanchez-Serrano JJ (1991) Abscisic acid mediates wound induction but not developmental-specific expression of the proteinase inhibitor II gene family. Plant Cell 3:963–972PubMedCrossRefGoogle Scholar
  44. Pena-Cortes H, Fisahn J, Willmitzer L (1995) Signals involved in wound-induced proteinase inhibitor II gene expression in tomato and potato plants. Proc Natl Acad Sci USA 92:4106–4113PubMedCrossRefGoogle Scholar
  45. Porta H, Rocha-Sosa M (2002) Plant lipoxygenases. Physiological and molecular features. Plant Physiol 130:15–21PubMedCrossRefGoogle Scholar
  46. Qu N, Schittko U, Baldwin IT (2004) Consistency of Nicotiana attenuata’s herbivore- and jasmonate-induced transcriptional responses in the allotetraploid species Nicotiana quadrivalvis and Nicotiana clevelandii. Plant Physiol 135:539–548PubMedCrossRefGoogle Scholar
  47. Schranz ME, Osborn TC (2000) Novel flowering time variation in the resynthesized polyploid Brassica napus. J Hered 91:242–246PubMedCrossRefGoogle Scholar
  48. Sidow A (1996) Gen(om)e duplications in the evolution of early vertebrates. Curr Opin Genet Dev 6:715–722PubMedCrossRefGoogle Scholar
  49. Soltis DE, Soltis PS (1995) The dynamic nature of polyploid genomes. Proc Natl Acad Sci USA 92:8089–8091PubMedCrossRefGoogle Scholar
  50. Soltis PS, Soltis DE (2000) The role of genetic and genomic attributes in the success of polyploids. Proc Natl Acad Sci USA 97:7051–7057PubMedCrossRefGoogle Scholar
  51. Soltis DE, Soltis PS, Tate JA (2004) Advances in the study of polyploidy since plant speciation. New Phytol 161:173–191CrossRefGoogle Scholar
  52. Song K, Lu P, Tang K, Osborn TC (1995) Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc Natl Acad Sci USA 92:7719–7723PubMedCrossRefGoogle Scholar
  53. The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815CrossRefGoogle Scholar
  54. Wang X, Shi X, Hao B, Ge S, Luo J (2005) Duplication and DNA segmental loss in the rice genome: implications for diploidization. New Phytol 165:937–946PubMedCrossRefGoogle Scholar
  55. Wendel JF (2000) Genome evolution in polyploids. Plant Mol Biol 42:225–249PubMedCrossRefGoogle Scholar
  56. Wolfe KH (2001) Yesterday’s polyploids and the mystery of diploidization. Nat Rev Genet 2:333–341PubMedCrossRefGoogle Scholar
  57. Zavala JA, Patankar AG, Gase K, Baldwin IT (2004a) Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata. Proc Natl Acad Sci USA 101:1607–1612CrossRefGoogle Scholar
  58. Zavala JA, Patankar AG, Gase K, Hui D, Baldwin IT (2004b) Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiol 134:1181–1190CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Jianqiang Wu
    • 1
  • Christian Hettenhausen
    • 1
  • Ian T. Baldwin
    • 1
  1. 1.Department of Molecular EcologyMax-Planck-Institute for Chemical EcologyJenaGermany

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