, Volume 219, Issue 3, pp 412–419 | Cite as

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate

  • Shai Koussevitzky
  • Emma Ne’eman
  • Eitan Harel
Original Article


Polyphenol oxidase (PPO; EC or EC takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8–16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.


Chloroplast import Jasmonic acid Lycopersicon Multicystatin Polyphenol oxidase Thylakoid translocation 




‘i’ and ‘p’

Prefixes used to denote the intermediate and precursor forms of a protein, respectively


Jasmonic acid


Large subunit of Rubisco


Methyl jasmonate

OE23 and OE33

23- and 33-kDa subunits of the oxygen-evolving complex of PSII




Precursor (intermediate, mature) form of polyphenol oxidase



This research was supported by grant US 2474-95C from The United States–Israel Binational Agricultural Research and Development Fund. We are extremely grateful to John C. Steffens, formerly of Cornell University, (Ithaca, NY) for providing the cloned ppo genes, and many helpful discussions. We thank Arieh Admon, the Protein Research Center, the Technion (Haifa, Israel) for protein sequencing, Abdussalam Azem, Biochemistry Department, Tel Aviv University (Tel Aviv, Israel) and Sophie Diamant, Department of Plant Sciences, The Hebrew University (Jerusalem, Israel) for help in the cross-linking experiments. We are grateful to Kunhua Chen, Todd Mockler and Todd Michael, Plant Biology laboratory, the Salk Institute (La Jolla, CA) for fruitful discussions and criticism.

This work is dedicated to Eitan Harel (1936–2003), a mentor and friend.


  1. Bolter CJ (1993) Methyl jasmonate induces papain inhibitor(s) in tomato leaves. Plant Physiol 103:1347–1353PubMedGoogle Scholar
  2. Boss PK, Gardner RC, Janssen BJ, Ross GS (1995) An apple polyphenol oxidase cDNA is up-regulated in wounded tissues. Plant Mol Biol 27:429–433PubMedGoogle Scholar
  3. Boulter D, Harvey PJ (1985) Accumulation, structure and utilization of tuber storage proteins, with particular reference to Dioscora rotunda. Physiol Veg 23:61–74Google Scholar
  4. Cheong JJ, Choi YD (2003) Methyl jasmonate as a vital substance in plants. Trends Gene 19:409–413CrossRefGoogle Scholar
  5. Chitnis PR, Nechushtai R, Thornber JP (1987) Insertion of the precursor of the light-harvesting chlorophyll a/b protein into the thylakoids requires the presence of a developmentally regulated stromal factor. Plant Mol Biol 10:3–11Google Scholar
  6. Constabel CP, Ryan CA (1998) A survey of wound- and methyl jasmonate-induced leaf polyphenol oxidase in crop plants. Phytochemistry 47:507–511CrossRefGoogle Scholar
  7. Constabel CP, Bergey BR, Ryan CA (1995) Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway. Proc Natl Acad Sci USA 92:407–411PubMedGoogle Scholar
  8. Creelman RA, Mullet JE (1997) Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol 48:355–381Google Scholar
  9. Dahlin C, Cline K (1991) Developmental regulation of the plastid protein import apparatus. Plant Cell 3:1131–1140CrossRefPubMedGoogle Scholar
  10. de Boer AD, Weisbeek PJ (1991) Chloroplast protein topogenesis: import, sorting and assembly. Biochim Biophys Acta 1071:221–253PubMedGoogle Scholar
  11. Duffy S, Felton G (1991) Enzymatic antinutritive defenses of the tomato plant against insects. In: Hedin P (ed) Naturally occurring pest bioregulators. American Chemical Society, Washington DC, pp 166–197Google Scholar
  12. Esterbauer H, Schwarzl E, Hayn M (1987) A rapid assay for catechol oxidase and laccase using 2-nitro 5-thiobenzoic acid. Anal Biochem 77:486–494Google Scholar
  13. Farmer EE, Ryan CA (1990) Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proc Natl Acad Sci USA 87:7713–7716PubMedGoogle Scholar
  14. Keegstra K, Cline K (1999) Protein import and routing systems in chloroplasts. Plant Cell 11:557–570PubMedGoogle Scholar
  15. Koussevitzky S, Ne’eman E. Sommer A, Steffens JC, Harel E (1998) Purification and properties of a novel chloroplast stromal peptidase. Processing of polyphenol oxidase and other imported precursors. J Biol Chem 273:27064–27069PubMedGoogle Scholar
  16. Kruzmane D, Jankevica L, Ievinsh G (2002) Effect of regurgitant from Leptinotarsa decemlineata on wound responses in Solanum tuberosum and Phaseolus vulgaris. Physiol Plant 115:577–584CrossRefPubMedGoogle Scholar
  17. Li l, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247CrossRefPubMedGoogle Scholar
  18. Mathew AG, Parpia HAB (1971) Food browning as a polyphenol reaction. Adv Food Res 19:75–145Google Scholar
  19. Mayer AM (1987) Polyphenol oxidases in plants—recent progress. Phytochemistry 26:11–20CrossRefGoogle Scholar
  20. Mayer AM, Harel E (1979) Polyphenol oxidases in plants. Phytochemistry 18:193–215Google Scholar
  21. Mayer AM, Harel E (1991) Phenoloxidases and their significance in fruit and vegetables. In: Fox PF (ed) Food enzymology, vol 1. Elsevier, New York, pp 373–398Google Scholar
  22. Newman SM, Eannetta NT, Yu H, Prince JP, De Vicente MC, Tanksley SD, Steffens JC (1993) Organization of the tomato polyphenol oxidase gene family. Plant Mol Biol 21:1035–1051PubMedGoogle Scholar
  23. 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 274:1914–1917PubMedGoogle Scholar
  24. Pieterse CMJ, van Wees SCM, van Pelt JA, Knoester M, Laan R, Gerrits H, Weisbeek PJ, van Loo LC (1998) A novel signaling pathway controlling induced systemic resistance in Arabidopsis. Plant Cell 10:1571–1580PubMedGoogle Scholar
  25. Ryan CA (2000) The systemin signaling pathway: differential activation of plant defensive genes. Biochim Biophys Acta 1477:112–121PubMedGoogle Scholar
  26. Reinbothe S, Mollenhauer B, Reinbothe C (1994) JIPs and RIPs: the regulation of plant gene expression by jasmonates in response to environmental cues and pathogens. Plant Cell 6:1197–1209CrossRefPubMedGoogle Scholar
  27. Rodis P, Hoff J (1984) Naturally occurring protein crystals in the potato. Inhibitor of papain, chymopapain, and ficin. Plant Physiol 74:907–911Google Scholar
  28. Rojo E, León J, Sánchez-Serrano J (1999) Cross-talk between wound signaling pathways determines local versus systemic gene expression in Arabidopsis thaliana. Plant J 20:135–142PubMedGoogle Scholar
  29. Schmelz EA, Hans TA, Tumlinson JH (2003) Synergistic interactions between volocitin, jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays. Physiol Plant 117:403–412CrossRefPubMedGoogle Scholar
  30. Sommer A, Ne’eman E, Steffens JC, Mayer AM, Harel E (1994) Import, targeting and processing of a plant polyphenol oxidase. Plant Physiol 105:1301–1311CrossRefPubMedGoogle Scholar
  31. Steffens JC, Harel E, Hunt MD (1994) Polyphenol oxidase. In: Ellis BE, Stafford HA (eds) Genetic engineering of plant secondary metabolism. Plenum, New York, pp 275–312Google Scholar
  32. Thipyapong P, Steffens JC (1997) Tomato polyphenol oxidase. Differential response of the polyphenol oxidase F promoter to injuries and wound signals. Plant Physiol 115:409–418Google Scholar
  33. Thipyapong P, Joel DM, Steffens JC (1997) Differential expression and turnover of the tomato polyphenol oxidase gene family during vegetative and reproductive development. Plant Physiol 113:707–718Google Scholar
  34. van Gelder CWG, Flurkey WH, Wichers HJ (1997) Sequence and structural features of plant and fungal tyrosinases. Phytochemistry 45:1309–1323CrossRefPubMedGoogle Scholar
  35. Vaughn KC, Lax AR, Duke SO (1988) Polyphenol oxidase: the chloroplast oxidase with no established function. Physiol Plant 72:659–665Google Scholar
  36. Waldron C, Wegrich P, Merlo AO, Walsh TA (1993) Characterization of a genomic sequence coding for potato multicystatin, an eight-domain cysteine proteinase inhibitor. Plant Mol Biol 23:801–812PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Shai Koussevitzky
    • 1
    • 2
  • Emma Ne’eman
    • 1
  • Eitan Harel
    • 1
  1. 1.Department of Plant SciencesThe Hebrew UniversityJerusalemIsrael
  2. 2.Plant Biology LaboratoryThe Salk Institute for Biological StudiesLa JollaUSA

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