, Volume 220, Issue 1, pp 87–96 | Cite as

Polyphenol oxidase overexpression in transgenic Populus enhances resistance to herbivory by forest tent caterpillar (Malacosoma disstria)

Original Article


In order to functionally analyze the predicted defensive role of leaf polyphenol oxidase (PPO; EC in Populus, transgenic hybrid aspen (Populus tremula × P. alba) plants overexpressing a hybrid poplar (Populus trichocarpa × P. deltoides) PtdPPO1 gene were constructed. Regenerated transgenic plants showed high PPO enzyme activity, PtdPPO1 mRNA levels and PPO protein accumulation. In leaf disk bioassays, forest tent caterpillar (Malacosoma disstria) larvae feeding on PPO-overexpressing transgenics experienced significantly higher mortality and reduced average weight gain compared to larvae feeding on control leaves. However, this effect was observed only when older egg masses were used and the resulting larvae showed reduced growth and vigor. In choice tests, no effect of PPO overexpression was detected. Although PPO in poplar leaves is latent and requires activation with detergents or trypsin for full enzymatic activity, in caterpillar frass the enzyme was extracted in the fully activated form. This activation correlated with partial proteolytic cleavage, suggesting that PPO latency and activation during digestion could be an adaptive and defense-related feature of poplar PPO.


Forest tent caterpillar Herbivore defense Plant–insect interaction Populus Polyphenol oxidase Transgenic aspen 



Cauliflower mosaic virus




Forest tent caterpillar




Leaf plastochron index


Polyphenol oxidase


Sodium dodecyl sulfate



The authors are grateful to Raju Datla (Plant Biotechnology Institute, Saskatoon, Canada) for providing the plant transformation vectors and host strains, Dave Ellis (CellFor Inc, Victoria, Canada) and Lise Jouanin (INRA, Versailles, France) for providing the hybrid aspen clone, Bob McCron (Canadian Forestry Service, Sault Ste Marie, Canada) for the forest tent caterpillar egg masses, and Brad Binges (Center for Forest Biology, University of Victoria) for help with plant maintenance. Funding was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada (C.P.C.) and by an FS Chia Fellowship from the University of Alberta (J.W.).


  1. Bergey DR, Howe GA, Ryan CA (1996) Polypeptide signaling for plant defensive genes exhibits analogies to defense signaling in animals. Proc Natl Acad Sci 93:12053–12058CrossRefPubMedGoogle Scholar
  2. Bradshaw HD Jr, Hollick JB, Parsons TJ, Clarke HR, Gordon MP (1990) Systemically wound-responsive genes in poplar trees encode proteins similar to sweet potato sporamins and legume Kunitz trypsin inhibitors. Plant Mol Biol 14:51–59PubMedGoogle Scholar
  3. Cho M-H, Moinuddin SGA, Helms GL, Hishiyama S, Eichinger D, Davin LB, Lewis NG (2003) (+)-Larreatricin hydroxylase, an enantio-specific polyphenol oxidase from the creosote bush (Larrea tridentata). Proc Natl Acad Sci USA 100:10641–10646CrossRefPubMedGoogle Scholar
  4. Christopher ME, Miranda M, Major IT, Constabel CP (2004) Gene expression profiling of systemically wound-induced defenses in hybrid poplar. Planta (in press)Google Scholar
  5. Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci 81:1991–1995PubMedGoogle Scholar
  6. Coetzer C, Corsini D, Love S, Pavek J, Tumer N (2001) Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase. J Agric Food Chem 49:652–657CrossRefPubMedGoogle Scholar
  7. Confalonieri M, Allegro G, Balestrazzi A, Fogher C, Delledonne M (1998) Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene. Mol Breed 4:137–145CrossRefGoogle Scholar
  8. Constabel CP, Ryan CA (1998) A survey of wound- and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry 47:507–511CrossRefGoogle Scholar
  9. Constabel CP, Bergey DR, Ryan CA (1996) Polyphenol oxidase as a component of the inducible defense response in tomato against herbivores. In: Romeo JT, Saunders JA, Barbosa P (eds) Phytochemical diversity and redundancy in ecological interactions. Plenum, New York, pp 231–252Google Scholar
  10. Constabel CP, Yip L, Patton JJ, Christopher ME (2000) Polyphenol oxidase from hybrid poplar. Cloning and expression in response to wounding and herbivory. Plant Physiol 124:285–295CrossRefPubMedGoogle Scholar
  11. Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 122:383–384CrossRefPubMedGoogle Scholar
  12. Datla RSS, Bekkaoui F, Hammerlindl JK, Pilate G, Dunstan DI, Crosby WL (1993) Improved high-level constitutive foreign gene expression in plants using an AMV RNA4 untranslated leader sequence. Plant Sci 94:139–149CrossRefGoogle Scholar
  13. Delledonne M, Allegro G, Belenghi B, Balestrazzi A, Picco F, Levine A, Zelasco S, Calligari P, Confalonieri M (2001) Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance. Mol Breed 7:35–42CrossRefGoogle Scholar
  14. Duffey SS, Felton GW (1991) Enzymatic antinutritive defenses of the tomato plant against insects. In: Hedin PA (ed) Naturally occurring pest bioregulators. ACS, Washington, DC, pp 167–197Google Scholar
  15. Ebling PM, Kaupp WJ (1997) Pathogenicity of a nuclear polyhedrosis virus to forest tent caterpillar, Malacosoma disstria (Hubner). Can Entomol 129:195–196Google Scholar
  16. Felton GW, Donato K, Delvecchio RJ, Duffey SS (1989) Activation of plant foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid herbivores. J Chem Ecol 15:2667–2694Google Scholar
  17. Felton GW, Donato KK, Broadway RM, Duffey SS (1992) Impact of oxidized plant phenolics on the nutritional quality of dietary protein to a noctuid herbivore, Spodoptera exigua. J Insect Physiol 38:277–285CrossRefGoogle Scholar
  18. Gill RI, Ellis BE, Isman MB (2003) Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores. J Chem Ecol 29:779–793CrossRefPubMedGoogle Scholar
  19. Haruta M, Pedersen JA, Constabel CP (2001a) Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates. Physiol Plant 112:552–558CrossRefPubMedGoogle Scholar
  20. Haruta M, Christopher ME, Patton JJ, Major IM, Constabel CP (2001b) A Kunitz trypsin inhibitor gene family from trembling aspen (Populus tremuloides Michx.): cloning, functional expression, and induction by wounding and herbivory. Plant Mol Biol 46:347–359CrossRefPubMedGoogle Scholar
  21. Humphreys JM, Chapple C (2002) Rewriting the lignin roadmap. Curr Opin Plant Biol 5:224–229CrossRefPubMedGoogle Scholar
  22. Kahn V, Andrawis A (1985) Inhibition of mushroom tyrosinase by tropolone. Phytochemistry 24:905–908CrossRefGoogle Scholar
  23. Kleiner KW, Ellis DD, McCown BH, Raffa KF (2003) Leaf ontogeny influences leaf phenolics and the efficacy of genetically expressed Bacillus thuringiensis cry1A(a) d-endotoxin in hybrid poplar against gypsy moth. J Chem Ecol 29:2585–2602CrossRefPubMedGoogle Scholar
  24. Koncz C, Schell J (1986) The promoter of TL DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396Google Scholar
  25. Kowalski SP, Eannetta NT, Hirzel AT, Steffens JC (1992) Purification and characterization of polyphenol oxidase from glandular trichomes of Solanum berthaultii. Plant Physiol 100:677–684Google Scholar
  26. Larson PR, Isebrands JG (1971) The plastochron index as applied to developmental studies of cottonwood. Can J For Res 1:1–11Google Scholar
  27. Leplé JC, Brasileiro ACM, Michel MF, Delmotte F, Jouanin L (1992) Transgenic poplars: expression of chimeric genes using four different constructs. Plant Cell Rep 11:137–141Google Scholar
  28. Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247CrossRefPubMedGoogle Scholar
  29. Mathew AG, Parpia HAB (1971) Food browning as a polyphenol reaction. Adv Food Res 19:75–145Google Scholar
  30. McCormac AC, Elliott MC, Chen DF (1998) A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation. Mol Biotechnol 9:155–159PubMedGoogle Scholar
  31. Murata M, Haruta M, Murai N, Tanikawa N, Nishimura M, Homma S, Itoh Y (2000) Transgenic apple (Malus × domestica) shoot showing low browning potential. J Agric Food Chem 48:5243–5248CrossRefPubMedGoogle Scholar
  32. Nakayama T, Yonekura-Sakakibara K, Sato T, Kikuchi S, Fukui Y, Fukuchi-Mizutani M, Ueda T, Nakao M, Tanaka Y, Kusumi T, Nishino T (2000) Aureusidin synthase: a polyphenol oxidase homolog responsible for flower coloration. Science 290:1163–1166CrossRefPubMedGoogle Scholar
  33. Parsons TJ, Bradshaw HD Jr, Gordon MP (1989) Systemic accumulation of specific mRNAs in response to wounding in poplar trees. Proc Natl Acad Sci USA 86:7895–7899PubMedGoogle Scholar
  34. Peters DJ, Constabel CP (2002) Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides). Plant J 32:701–712CrossRefPubMedGoogle Scholar
  35. Robinson SP, Dry IB (1992) Broad bean leaf polyphenol oxidase is a 60-kilodalton protein susceptible to proteolytic cleavage. Plant Physiol 99:317–323Google Scholar
  36. Robison DJ, McCown BH, Raffa KF (1994) Responses of gypsy moth (Lepidoptera, Lymantriidae) and forest tent caterpillar (Malacosoma disstria) to transgenic poplar, Populus spp containing a Bacillus thuringiensis D-endotoxin gene. Environ Entomol 23:1030–1041Google Scholar
  37. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
  38. Singleton VL, Rossi JA (1965) Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Vitic 16:144–158Google Scholar
  39. Steffens JC, Harel E, Hunt MD (1994) Polyphenol oxidase. In: Ellis BE, Kuroki GW, Stafford HA (eds) Genetic engineering of plant secondary metabolism. Plenum, New York, pp 276–304Google Scholar
  40. Vaughn KC, Duke SO (1984) Function of polyphenol oxidase in higher plants. Physiol Plant 60:106–112Google Scholar
  41. Wang J, Constabel CP (2003) Biochemical characterization of two differentially expressed polyphenol oxidases from hybrid poplar. Phytochemistry 64:115–121CrossRefPubMedGoogle Scholar
  42. Wang J, Constabel CP (2004) Three Polyphenol Oxidases from Hybrid Poplar Are Differentially Expressed During Development and After Wounding and Elicitor Treatment. Physiol Plant (in press)Google Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Centre for Forest Biology and Department of BiologyUniversity of VictoriaVictoriaCanada
  2. 2.Department of Forest Genetics and Plant PhysiologySwedish University of Agricultural SciencesUmeaSweden

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