Plant Growth Regulation

, Volume 71, Issue 3, pp 225–233 | Cite as

Polyphenol gene expression and changes in anthocyanins and polyphenols in the skin of ‘Braeburn’ apples after the autumn application of prohexadione-calcium

  • Jan BizjakEmail author
  • Nika Weber
  • Maja Mikulic-Petkovsek
  • Zobayer Alam
  • Jana Thill
  • Karl Stich
  • Heidi Halbwirth
  • Robert Veberic
Original paper


Prohexadione-calcium (Pro-Ca) transient inhibits 2-Oxoglutarate-dependent dioxygenases and causes significant changes in the flavonoid spectrum of apple. In the present study the influence of two autumn preharvest applications of Pro-Ca on the polyphenol metabolism in apple peel during the advanced maturation was investigated. Pro-Ca was sprayed in two doses, approximately five and 3 weeks before the technological maturity. Changes in the concentrations of hydroxycinnamic acids, dihydrochalcones, flavonols, flavanols and anthocyanins as well as their related gene expression and enzyme activities in the apple peel were monitored six times during the advanced maturation until the technological maturity of the fruits. To evaluate its influence on red coloration differences in the chromatic values a*, h° and L* between the treated and untreated apples were monitored. The parameters showed a temporary effect of Pro-Ca on the intensity of red coloration, which was not detected anymore at the technological maturity of apples. The application of Pro-Ca decreased the flavanone 3-hydroxylase activity and slightly inhibited activities of all the enzymes analyzed. Concomitantly, the concentrations of anthocyanins in the peel of the treated apples decreased, whereas the concentrations of hydroxycinnamic acids, dihydrochalcones and flavan 3-ols increased. Flavonol concentrations, however, remained unchanged. The expression of ANS, ANR, FGT and MYB10 was downregulated after the Pro-Ca treatment. The results indicate that the autumn application of Pro-Ca modulates the biosynthetic pathway resulting in distinct changes in the flavonoid composition in the apple peel of ‘Braeburn’ apples. However, the changes are temporary and are generally suspended during apple storage.


Flavonoids Anthocyanins Gene expression Phenolic acids Prohexadione-calcium 







Fresh weight


Phenylalanine ammonia-lyase


Chalcone synthase/chalcone isomerase


Flavanone 3-hydroxylase


Dihydroflavonol 4-reductase


Anthocyanidin synthase


Anthocyanidin reductase


Flavonoid 3-O-glycosyltransferase


Elongation factor



This investigation was part of the Horticulture Program P4-0013-0481 and the authors would like to thank the Slovenian Ministry of Higher Education, Science and Technology for the financial support. The work was performed within the frame of PlantEngine (Cost Action FA1006).


  1. Bazzi C, Messina C, Tortoreto L, Bini F, Cecca GS, Stefani E (2003) Investigations on the possible use of abiotic and biotic elicitors in defence-related responses in plants. Eur J Hortic Sci 68:115–122Google Scholar
  2. Bizjak J, Jakopic J, Slatnar A, Stampar F, Stich K, Halbwirth H, Zadravec P, Veberic R (2012) Late prohexadione-calcium application on maturing apple cv. ‘Braeburn’ fruit reduces anthocyanins and alters the phenolic content. Eur J Hortic Sci 77:154–162Google Scholar
  3. Chang S, Pur Year J, Carney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  4. Fischer TC, Halbwirth H, Römmelt S, Sabatini E, Schlangen K, Andreotti C, Spinelli F, Costa G, Forkmann G, Treutter D, Stich K (2006) Induction of flavonoid gene expression in apple (Malus x domestica) after the application of a dioxygenase enzyme inhibitor. Physiol Plant 128:604–617CrossRefGoogle Scholar
  5. Gosch C, Puhl I, Halbwirth H, Schlangen K, Roemmelt S, Andreotti C, Costa G, Fischer TC, Treutter D, Stich K, Forkmann G (2003) Effect of prohexadione-Ca on various fruit crops: flavonoid composition and substrate specificy of their dihydroflavonol 4-reductases. Eur J Hortic Sci 68:144–151Google Scholar
  6. Halbwirth H, Fischer TC, Roemmelt S, Spinelli F, Schlangen K, Peterek S, Sabatini E, Messina C, Speakman J-B, Andreotti C, Rademacher W, Bazzi C, Costa G, Treutter D, Forkmann G, Stich K (2003) Induction of antimicrobial 3-deoxyflavonoids in pome fruit trees controls fire blight. Zeitschr Naturf 58c:765–770Google Scholar
  7. Halbwirth H, Fischer TC, Schlangen K, Rademacher W, Schleifer K-J, Forkmann G, Stich K (2006) Screening for inhibitors of 2-oxoglutarate-dependent dioxygenases: flavanone 3β-hydroxylase and flavonol synthase. Plant Sci 171:194–205CrossRefGoogle Scholar
  8. Honda C, Kotoda N, Wada M, Kondo S, Kobayashi S, Soejima J, Zhang Z, Tsuda T, Moriguchi T (2002) Anthocyanin biosynthetic genes are coordinately expressed during red coloration in apple skin. Plant Physiol Biochem 40:955–962CrossRefGoogle Scholar
  9. Krawczyk G, Greene GM (2002) The impact of plant growth regulator apogee on insect pest populations and fruit quality. PA Fruit News 82:18–24Google Scholar
  10. Marks SC, Mullen W, Crozier A (2007) Flavonoid and chlorogenic acid profiles of English cider apples. J Sci Food Agric 87:719–728CrossRefGoogle Scholar
  11. McGuire RG (1992) Reporting objective colour measurements. HortScience 27:1254–1255Google Scholar
  12. Medjdoub R, Val J, Blanco A (2005) Inhibition of vegetative growth in red apple cultivars using prohexadione-calcium. J Hortic Sci Biotech 80:263–271Google Scholar
  13. Mikulic-Petkovsek M, Štampar F, Veberic R (2009) The effect of prohexadione-calcium content in developing fruits and leaves of apple trees. J Food Agric Environ 7:369–375Google Scholar
  14. Mikulic-Petkovsek M, Slatnar A, Stampar F, Veberic R (2010) The influence of organic/integrated production on the content of phenolic compounds in apple leaves and fruits in four different varieties over a 2-year period. J Sci Food Agric 90:2366–2378PubMedCrossRefGoogle Scholar
  15. Rademacher W, Kober R (2003) Efficient use of prohexadione-Ca in pome fruits. Eur J Hortic Sci 68:101–107Google Scholar
  16. Rademacher W, Temple–Smith KE, Griggs DL, Hedden P (1992) The mode of action of acylcyclohexanediones-a new type of growth retardant. Curr Plant Sci Biotechnol Agric 13:571–577CrossRefGoogle Scholar
  17. Roemmelt S, Zimmermann N, Rademacher W, Treutter D (2003) Formation of novel flavonoids in apple (Malus x domestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca. Phytochem 64:709–716CrossRefGoogle Scholar
  18. Slatnar A, Mikulic-Petkovsek M, Halbwirth H, Stampar F, Stich K, Veberic R (2012) Polyphenol metabolism of developing apple skin of a scab resistant and a susceptible apple cultivar. Trees 26:109–119CrossRefGoogle Scholar
  19. Spinelli F, Speakman JB, Rademacher W, Halbwirth H, Stich K, Costa G (2005) Luteoforol, a flavan 4-ol, is induced in pome fruits by prohexadione-Ca and shows phytoalexin-like properties against Erwinia amylovora and other plant pathogens. Eur J Plant Pathol 111:1–10CrossRefGoogle Scholar
  20. Takos AM, Jaffé FW, Jacob SR, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142:1216–1232PubMedCrossRefGoogle Scholar
  21. Telias A, Wang KL, Stevenson DE, Cooney JM, Hellens RP, Allan AC, Hoover EE, Bradeen JM (2011) Apple skin patterning is associated with differential expression of MYB10. BMC Plant Biol 11:93PubMedCrossRefGoogle Scholar
  22. Tsao R, Yang R, Xie S, Sockovie E, Khanizadeh S (2005) Which polyphenolic compounds contribute to the total antioxidant activities of apple? J Agric Food Chem 53:4989–4995PubMedCrossRefGoogle Scholar
  23. Yoder KS, Miller SS, Byers RE (1999) Suppression of fire blight in apple shoots by prohexadione-calcium following experimental and natural inoculation. HortScience 34:1202–1204Google Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Jan Bizjak
    • 1
    Email author
  • Nika Weber
    • 1
  • Maja Mikulic-Petkovsek
    • 1
  • Zobayer Alam
    • 2
  • Jana Thill
    • 2
  • Karl Stich
    • 2
  • Heidi Halbwirth
    • 2
  • Robert Veberic
    • 1
  1. 1.Department of Agronomy, Biotechnical Faculty, Chair for Fruit, Wine and Vegetable GrowingUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Institute for Chemical EngineeringTechnical University of ViennaViennaAustria

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