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Planta

, Volume 220, Issue 1, pp 105–117 | Cite as

Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility

  • Piyada ThipyapongEmail author
  • Michelle D. Hunt
  • John C. Steffens
Original Article

Abstract

Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm−2 than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm−2 than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::β-glucuronidase constructs when plants are challenged with P. syringae in both Pto and pto backgrounds. While PPO B inducibility was the same in both compatible and incompatible interactions, PPO D, E and F were induced to higher levels and with different expression patterns in incompatible interactions.

Keywords

Antisense Disease resistance Lycopersicon Polyphenol oxidase Pseudomonas 

Abbreviations

DOPA

3,4-Dihydroxyphenylalanine

NT

Nontransformed

PO

Peroxidase

PPO

Polyphenol oxidase

ROS

Reactive oxygen species

SA

Salicylic acid

Notes

Acknowledgements

We are grateful to Dr. S.M. Newman for providing the transgenic tomato carrying the PPO A, B, D, E and F promoter::GUS constructs. We also thank Drs. S.D. Tanksley and A. Frary for providing P. syringae culture and the inoculation procedure, Dr. E.D. Earle for the use of microscope facilities, and C. Dougherty for the transmission electron microscope work. This work was supported by grants from the United States Department of Agriculture National Research Initiative (grant no. 91-37301-6571) and the Binational Agricultural Research and Development Fund (grant no. US 1870-90) to J.C.S.

References

  1. Bachem CWB, Speckmann GJ, van der Linde PCG, Verheggen FTM, Hunt MD, Steffens JC, Zabeau M (1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Biotechnology 12:1101–1105Google Scholar
  2. Bashan Y (1986) Phenols in cotton seedlings resistant and susceptible to Alternaria macrospora. J Phytopathol 116:1–10Google Scholar
  3. Bashan Y, Okon Y, Henis Y (1987) Peroxidase, polyphenoloxidase, and phenols in relation to resistance against Pseudomonas syringae pv. tomato in tomato plants. Can J Bot 65:366–372Google Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. 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
  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 DR, 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. Felton GW, Donato K, Del Vecchio 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
  9. Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chapple C (2002) The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J 30:33–45CrossRefPubMedGoogle Scholar
  10. Friedman M (1997) Chemistry, biochemistry, and dietary role of potato polyphenols. J Agric Food Chem 45:1523–1540CrossRefGoogle Scholar
  11. Garcia-Olmedo F, Rodriguez-Palenzuela P, Molina A, Alamillo JM, Lopez-Solanilla E, Berrocal-Lobo M, Poza-Carrion C (2001) Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence. FEBS Lett 498:219–222CrossRefPubMedGoogle Scholar
  12. Grant JJ, Loake GJ (2000) Role of reactive oxygen intermediates and cognate redox signaling in disease resistance. Plant Physiol 124:21–29CrossRefPubMedGoogle Scholar
  13. Guyot S, Cheynier V, Souquet JM, Moutounet M (1995) Influence of pH on the enzymatic oxidation of (+)-catechin in model systems. J Agric Food Chem 43:2458–2462Google Scholar
  14. Guyot S, Vercauteren J, Cheynier V (1996) Structural determination of colourless and yellow dimers resulting from (+)-catechin coupling catalysed by grape polyphenol oxidase. Phytochemistry 42:1279–1288CrossRefGoogle Scholar
  15. Hammett LP (1970) Physical organic chemistry; reaction rates, equilibria, and mechanisms, 2nd edn. McGraw-Hill, New YorkGoogle Scholar
  16. Haruta M, Pedersen JA, Constabel CP (2001) Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates. Physiol Plant 112:552–558CrossRefPubMedGoogle Scholar
  17. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180Google Scholar
  18. Hunt MD, Eannetta NT, Yu H, Newman SM, Steffens JC (1993) cDNA cloning and expression of potato polyphenol oxidase. Plant Mol Biol 21:59–68PubMedGoogle Scholar
  19. Jiang Y, Miles PW (1993) Generation of H2O2 during enzymatic oxidation of catechin. Phytochemistry 33:29–34CrossRefGoogle Scholar
  20. Khirbat SK, Jalali BL (1998) Polyphenoloxidase and bound phenol content in the leaves of chickpea (Cicer arietinum L.) after inoculation with Ascochyta rabiei. Legume Res 21:198–200Google Scholar
  21. Kojima M, Takeuchi W (1989) Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings. J Biochem 105:265–270PubMedGoogle Scholar
  22. Kowalski SP, Plaisted RL, Steffens JC (1993) Immunodetection of polyphenol oxidase in glandular trichomes of Solanum berthaultii, Solanum tuberosum and their hybrids. Am Potato J 70:185–199Google Scholar
  23. Levine A, Pennell RI, Alvarez ME, Palmer R, Lamb C (1996) Calcium-mediated apoptosis in a plant hypersensitive disease resistance response. Curr Biol 6:427–437CrossRefPubMedGoogle Scholar
  24. Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215:239–247CrossRefPubMedGoogle Scholar
  25. Maher EA, Bate NJ, Ni W, Elkind Y, Dixon RA (1994) Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products. Proc Natl Acad Sci USA 91:7802–7806Google Scholar
  26. Martin GB, Brommonschenkel SH, Chunwongse J, Frary A, Ganal MW, Spivey R, Wu T, Earle ED, Tanksley SD (1993) Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262:1432–1436PubMedGoogle Scholar
  27. Mayer AM, Harel E (1991) Phenoloxidases and their significance in fruit and vegetables. In: Fox PF (ed) Food enzymology. Elsevier, New York, pp 373–398Google Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497Google Scholar
  29. Newman SM, Eannetta NT, Yu H, Prince J, Tanksley SD, Steffens JC (1993) Characterization of the tomato polyphenol oxidase gene family. Plant Mol Biol 21:1035–1052PubMedGoogle Scholar
  30. Orozco-Cárdenas ML, Narvaez-Vasquez J, Ryan CA (2001) Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin, and methyl jasmonate. Plant Cell 13:179–191CrossRefPubMedGoogle Scholar
  31. Peter MG (1989) Chemical modifications of biopolymers by quinones and quinone methides. Angew Chem Int Ed Engl 28:555–570CrossRefGoogle Scholar
  32. Richard-Forget FC, Gauillard FA (1997) Oxidation of chlorogenic acid, catechins, and 4-methylcatechol in model solutions by combinations of pear (Pyrus communis cv. Williams) polyphenol oxidase and peroxidase: a possible involvement of peroxidase in enzymatic browning. J Agric Food Chem 45:2472–2476CrossRefGoogle Scholar
  33. Ronald PC, Salmeron JM, Carland FM, Staskawicz BJ (1992) The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene. J Bacteriol 174:1604–1611PubMedGoogle Scholar
  34. Shahar T, Hennig N, Gutfinger T, Hareven D, Lifschitz E (1992) The tomato 66.3-kD polyphenol oxidase gene: molecular identification and developmental expression. Plant Cell 4:135–147CrossRefPubMedGoogle Scholar
  35. Sherman TD, Vaughn KC, Duke SO (1991) A limited survey of the phylogenetic distribution of polyphenol oxidase. Phytochemistry 30:2499–2506CrossRefGoogle Scholar
  36. Sommer A, Néeman E, Steffens JC, Mayer AM, Harel E (1994) Import, targeting and processing of a plant polyphenol oxidase. Plant Physiol 105:1301–1311Google Scholar
  37. Stout MJ, Workman KV, Bostock RM, Duffey SS (1998) Stimulation and attenuation of induced resistance by elicitors and inhibitors of chemical induction in tomato (Lycopersicon esculentum) foliage. Entomol Exp Appl 86:267–279Google Scholar
  38. Thipyapong P, Steffens JC (1997) Tomato polyphenol oxidase: Differential response of the polyphenol oxidase F promoter to injuries and wound signals. Plant Physiol 115:409–418PubMedGoogle Scholar
  39. Thipyapong P, Hunt MD, Steffens JC (1995) Systemic wound induction of potato (Solanum tuberosum) polyphenol oxidase. Phytochemistry 40:673–676CrossRefGoogle Scholar
  40. 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 113:707–718PubMedGoogle Scholar
  41. Thipyapong P, Melkonian J, Wolfe DW, Steffens JC (2004) Suppression of polyphenol oxidases increases stress tolerance in tomato. Plant Sci 167:693–703CrossRefGoogle Scholar
  42. Torres MA, Dangl JL, Jones DG (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522CrossRefPubMedGoogle Scholar
  43. Trebst A, Depka B (1995) Polyphenol oxidase and photosynthesis research. Photosynth Res 46:41–44Google Scholar
  44. Vaughn KC, Duke SO (1981) Tissue localization of polyphenol oxidase in sorghum. Protoplasma 108:319–327Google Scholar
  45. Vaughn KC, Duke SO (1982) Tentoxin effects on sorghum: the role of polyphenol oxidase. Protoplasma 110:48–53Google Scholar
  46. Vaughn KC, Duke SO (1984) Function of polyphenol oxidase in higher plants. Physiol Plant 60:106–112Google Scholar
  47. Vaughn KC, Lax AR, Duke SO (1988) Polyphenol oxidase: the chloroplast oxidase with no established function. Physiol Plant 72:659–665Google Scholar
  48. Yao K, De Luca V, Brisson N (1995) Creation of a metabolic sink for tryptophan alters the phenylpropanoid pathway and the susceptibility of potato to Phytophthora infestans. Plant Cell 7:1787–1799CrossRefPubMedGoogle Scholar
  49. Zhou J, Tang X, Martin GB (1997) The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind cis-element of pathogenesis-related genes. EMBO J 16:3207–3218CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Piyada Thipyapong
    • 1
    • 2
    Email author
  • Michelle D. Hunt
    • 1
    • 3
  • John C. Steffens
    • 1
    • 4
  1. 1.Department of Plant BreedingCornell UniversityIthacaUSA
  2. 2.Suranaree University of TechnologyNakhon RatchasimaThailand
  3. 3.CropsolutionResearch Triangle ParkUSA
  4. 4.Syngenta BiotechnologyResearch Triangle ParkUSA

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