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Planta

, Volume 226, Issue 5, pp 1243–1254 | Cite as

Maize Lc transcription factor enhances biosynthesis of anthocyanins, distinct proanthocyanidins and phenylpropanoids in apple (Malus domestica Borkh.)

  • Houhua Li
  • Henryk Flachowsky
  • Thilo C. Fischer
  • Magda-Viola Hanke
  • Gert Forkmann
  • Dieter Treutter
  • Wilfried Schwab
  • Thomas Hoffmann
  • Iris SzankowskiEmail author
Original Article

Abstract

Flavonoids are a large family of polyphenolic compounds with manifold functions in plants. Present in a wide range of vegetables and fruits, flavonoids form an integral part of the human diet and confer multiple health benefits. Here, we report on metabolic engineering of the flavonoid biosynthetic pathways in apple (Malus domestica Borkh.) by overexpression of the maize (Zea mays L.) leaf colour (Lc) regulatory gene. The Lc gene was transferred into the M. domestica cultivar Holsteiner Cox via Agrobacterium tumefaciens-mediated transformation which resulted in enhanced anthocyanin accumulation in regenerated shoots. Five independent Lc lines were investigated for integration of Lc into the plant genome by Southern blot and PCR analyses. The Lc-transgenic lines contained one or two Lc gene copies and showed increased mRNA levels for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), flavanone 3 beta-hydroxylase (FHT), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductases (LAR), anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). HPLC-DAD and LC-MS analyses revealed higher levels of the anthocyanin idaein (12-fold), the flavan 3-ol epicatechin (14-fold), and especially the isomeric catechin (41-fold), and some distinct dimeric proanthocyanidins (7 to 134-fold) in leaf tissues of Lc-transgenic lines. The levels of phenylpropanoids and their derivatives were only slightly increased. Thus, Lc overexpression in Malus domestica resulted in enhanced biosynthesis of specific flavonoid classes, which play important roles in both phytopathology and human health.

Keywords

Agrobacterium Flavonoids Metabolic Engineering Transformation 

Abbreviations

ANS

Anthocyanidin synthase

ANR

Anthocyanidin reductase

BAP

6-Benzylaminopurine

CaMV

Cauliflower mosaic virus

CHI

Chalcone isomerase

CHS

Chalcone synthase

DAD

Diode array detection

DFR

Dihydroflavonol 4-reductase

FGT

UDP-Glucose:flavonoid 3-O-glucosyltransferase

FHT

Flavanon 3 beta-hydroxylase

FLS

Flavonol synthase

GA

Gibberellic acid

HPLC

High performance liquid chromatography

HC

Holsteiner Cox

IBA

Indole-3-butyric acid

LAR1+2

Leucoanthocyanidin reductase

Lc

Maize leaf colour

LC-MS

Liquid chromatography/mass spectroscopy

MS

Murashige and Skoog

PAL

Phenylalanin ammonia-lyase

TDZ

Thidiazuron

YEP

Yeast extract broth

Notes

Acknowledgements

We thank Ryan Peeler and Sue Wessler (Plant Biology Department, University of Georgia, Athens, GA, USA) for providing the Lc-gene. This work was partially supported by the Ministry of Science and Culture (MWK) of the state Lower Saxony and the Federal Ministry for Education and Research (BMBF, project number 0312638C), Germany.

References

  1. Andreotti C, Costa G, Treutter D (2006) Composition of phenolic in pear leaves as affected by genetics, ontogenesis and the environment. Sci Hortic 109:130–137CrossRefGoogle Scholar
  2. Bazzi C, Messina C, Tortoreto L, Stefani E, Bini F, Brunelli A, Andreotti C, Sabatini E, Spinelli F, Costa G, Hauptmann S, Stammler G, Doerr S, Marr J, Rademacher W (2003) Control of pathogen incidence in pome fruits and other horticultural crop plants with prohexadione-Ca. Eur J Hortic Sci 68:108–114Google Scholar
  3. Bovy A, de Vos R, Kemper M, Schijlen E, Almenar Pertejo M, Muir S, Collins G, Robinson S, Verhoeyen M, Hughes S, Santos-Buelga C, van Tunen A (2002) High-flavonol tomatoes resulting from the heterologous expression of the maize transcription factor genes LC and C1. Plant Cell 14:2509–2526PubMedCrossRefGoogle Scholar
  4. Bradley JM, Davies KM, Deroles SC, Bloor SJ, Lewis DH (1998) The maize Lc regulatory gene up-regulates the flavonoid biosynthetic pathway of Petunia. Plant J 13:381–392CrossRefGoogle Scholar
  5. Byrne PF, McMullen MD, Snook ME, Musket TA, Theuri JM, Widstrom NW, Wiseman BR, Coe EH Jr (1996) Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks. Proc Natl Acad Sci USA 93:8820–8825PubMedCrossRefGoogle Scholar
  6. Challice JS, Williams AH (1968) Phenolic compounds of the genus Pyrus—II. A chemotaxonomic survey. Phytochemistry 7:1781–1801CrossRefGoogle Scholar
  7. Commenges D, Scotet V, Renaud S, Jacqmin-Gadda H, Barberger-Gateau P, Dartigues JF (2000) Intake of flavonoids and risk of dementia. Eur J Epidemiol 16:357–363PubMedCrossRefGoogle Scholar
  8. Cushman M, Nagarathnam D, Burg DL, Geahlen RL (1991) Synthesis and protein-tyrosine kinase inhibitory activities of flavonoid analogues. J Med Chem 34:798–806PubMedCrossRefGoogle Scholar
  9. Degenhardt J, Poppe A, Montag J, Szankowski I (2006) The use of the phosphomannose-isomerase/mannose selection system to recover transgenic apple plants. Plant Cell Rep 25:1149–1156PubMedCrossRefGoogle Scholar
  10. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  11. Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2006) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427PubMedCrossRefGoogle Scholar
  12. Ford CM, Boss PK, Høj PB (1998) Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 Locus that may primarily serve to glucosylate anthocyanidins in vivo. J Biol Chem 273:9224–9233PubMedCrossRefGoogle Scholar
  13. Geahlen RL, Koonchanok NM, McLaughlin JL, Pratt DE (1989) Inhibition of protein-tyrosine kinase activity by flavonoids and related compounds. J Nat Prod 52:982–986PubMedCrossRefGoogle Scholar
  14. Goldsbrough AP, Tong Y, Yoder JI (1996) Lc as a non-destructive visual reporter and transposition excision marker gene for tomato. Plant J 9:927–933CrossRefGoogle Scholar
  15. Grotewold E (2005) Plant metabolic diversity: a regulatory perspective. Trends Plant Sci 10:57–62PubMedCrossRefGoogle Scholar
  16. Halbwirth H, Fischer TC, Römmelt S, Spinelli F, Schlangen K, Peterek S, Sabatini E, Messina C, Speakman JB, 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. Z Naturforsch 58c:765–770Google Scholar
  17. Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:735–747PubMedCrossRefGoogle Scholar
  18. Hertog MGL, Feskens EJ, Kromhout D (1997) Antioxidant flavonols and coronary heart disease risk. Lancet 349:699PubMedCrossRefGoogle Scholar
  19. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium vectors for plant transformation. Transgenic Res 2:208–218CrossRefGoogle Scholar
  20. Jambunathan R, Kherdekar MS, Bandyopadhay R (1990) Flavan 4-ols concentration in mold susceptible and mold resistant Sorghum at different stages of grain development. J Agric Food Chem 38:545CrossRefGoogle Scholar
  21. Katajamaa M, Miettinen J, Oresic M (2006) MZmine: toolbox for processing and visualization of mass spectrometry based molecular profile data. Bioinformatics 22:634–636PubMedCrossRefGoogle Scholar
  22. Knekt P, Isotupa S, Rissanen H, Heliovaara M, Jarvinen R, Hakkinen S, Aromaa A, Reunanen A (2000) Quercetin intake and the incidence of cerebrovascular disease. Eur J Clin Nutr 54:415–417PubMedCrossRefGoogle Scholar
  23. Knekt P, Järvinen R, Seppänen R, Heliövaara M, Teppo L, Pukkala E, Aromaa A (1997) Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol 146:223–230PubMedGoogle Scholar
  24. Lattanzio V, Cardinali A, Palmieri S (1994) The role of phenolics in the postharvest physiology of fruits and vegetables: browning reactions and fungal diseases. Ital J Food Sci 1:3–22Google Scholar
  25. Lattanzio V (2003) Bioactive polyphenols: their role in quality and storability of fruit and vegetables. J Appl Bot 77:128–146Google Scholar
  26. Leser C, Treutter D (2005) Effects of nitrogen supply on growth, contents of phenolic compounds and pathogen (scab) resistance of apple trees. Physiol Plant 123:49–56CrossRefGoogle Scholar
  27. Li SJ, Deng XM, Mao HZ, Hong Y (2005) Enhanced anthocyanin synthesis in foliage plant Caladium bicolor. Plant Cell Rep 23:716–720PubMedCrossRefGoogle Scholar
  28. Lloyd AM, Walbot V, Davis RW (1992) Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258:1773–1775PubMedCrossRefGoogle Scholar
  29. Ludwig SR, Habera LF, Dellaporta SL, Wessler SR (1989) Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region. Proc Natl Acad Sci USA 86:7092–7096PubMedCrossRefGoogle Scholar
  30. Mayr U, Treutter D, Santos-Buelga C, Bauer H, Feucht W (1995) Developmental changes in the phenol concentrations of ‘Golden delicious’ apple fruits and leaves. Phytochemistry 38:1151–1155PubMedCrossRefGoogle Scholar
  31. Mayr U, Michalek S, Treutter D, Feucht W (1997) Phenolic compounds of apple and their relationship to scab resistance. J Phytopathol 145:69–75Google Scholar
  32. Middleton E Jr, Kandaswami C, Theoharides TC (2000) The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol Rev 52:673–751PubMedGoogle Scholar
  33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497CrossRefGoogle Scholar
  34. Quattrocchio F, Wing JF, Leppen HTC, Mol JNM, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell 5:1497–1512PubMedCrossRefGoogle Scholar
  35. Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F (2007) Ectopic Expression of a basic helix-loop-helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase genes in Lotus corniculatus. Plant Physiol 143:504–516PubMedCrossRefGoogle Scholar
  36. Ray H, Yu M, Auser P, Blahut-Beatty L, McKersie B, Bowley S, Westcott N, Coulman B, Lloyd A, Gruber MY (2003) Expression of anthocyanins and proanthocyanidins after transformation of alfalfa with maize Lc. Plant Physiol 132:1448–1463PubMedCrossRefGoogle Scholar
  37. Römmelt S, Zimmermann N, Rademacher W, Treutter D (2003a) Formation of novel flavonoids in apple (Malus domestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca. Phytochemistry 64:709–716CrossRefGoogle Scholar
  38. Römmelt S, Fischer TC, Halbwirth H, Peterek S, Schlangen K, Speakman JB, Treutter D, Forkmann G, Stich K (2003b) Effect of dioxygenase inhibitors on the resistance-related flavonoid metabolism of apple and pears: chemical, biochemical and molecular biological aspects. Eur J Hortic Sci 68:129–136Google Scholar
  39. Römmelt S, Treutter D, Speakman JB, Rademacher W (1999) Effects of prohexadione-Ca on the flavonoid metabolism of apple with respect to plant resistance against fire blight. Acta Hortic 489:359–363Google Scholar
  40. Rühmann S, Treutter D, Fritsche S, Briviba K, Szankowski I (2006) Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit. J Agric Food Chem 54:4633–4640PubMedCrossRefGoogle Scholar
  41. Szankowski I, Briviba K, Fleschhut J, Schönherr J, Jacobsen HJ, Kiesecker H (2003) Transformation of apple (Malus domestica Borkh.) with the stilbene synthase gene from grapevine (Vitis vinifera L.) and a PGIP gene from kiwi (Actinidia deliciosa). Plant Cell Rep 22:141–149PubMedCrossRefGoogle Scholar
  42. Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol. doi:10.1104/pp.106.088104Google Scholar
  43. Taylor LP, Grotewold E (2005) Flavonoids as developmental regulators. Curr Opin Plant Biol 8:317–323PubMedCrossRefGoogle Scholar
  44. Treutter D (1989) Chemical reaction detection of catechins and proanthocyanidins with 4-dimethylaminocinnamaldehyde. J Chromatogr A 467:185–193CrossRefGoogle Scholar
  45. Treutter D, Santos-Buelga C, Gutmann M, Kolodziej H (1994) Identification of flavan-3-ols and procyanidins by highperformance liquid chromatography and chemical reaction detection. J Chromatogr A 667:290–297CrossRefGoogle Scholar
  46. Treutter D (2001) Biosynthesis of phenolic compounds and its regulation in apple. Plant Growth Regul 34:71–89CrossRefGoogle Scholar
  47. Treutter D (2005) Significance of flavonoids in plant resistance and enhancement of their biosynthesis. Plant Biol 7:581–591PubMedCrossRefGoogle Scholar
  48. Treutter D, Feucht W (1990) The pattern of flavan-3-ols in relation to scab resistance. J Hortic Sci 65:511–517Google Scholar
  49. 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
  50. Vandesompele J, De PK, Pattyn F, Poppe B, Van RN, De PA, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:1–12Google Scholar
  51. Wellmann F, Griesser M, Schwab W, Martens S, Eisenreich W, Matern U, Lukačin R (2006) Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin. FEBS Letters 580:1642–1648PubMedCrossRefGoogle Scholar
  52. Williams RJ, Spencer JPE, Rice-Evans C (2004) Flavonoids: antioxidants or signalling molecules? Free Radical Biol Med 36:838–849CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Houhua Li
    • 1
  • Henryk Flachowsky
    • 2
  • Thilo C. Fischer
    • 3
  • Magda-Viola Hanke
    • 2
  • Gert Forkmann
    • 3
  • Dieter Treutter
    • 4
  • Wilfried Schwab
    • 5
  • Thomas Hoffmann
    • 5
  • Iris Szankowski
    • 1
    Email author
  1. 1.Institute of Biological Production Systems, Fruit Science SectionLeibniz University of HannoverHannoverGermany
  2. 2.Federal Centre for Breeding Research on Cultivated Plants, Institute of Fruit BreedingDresdenGermany
  3. 3.Chair for Ornamental Plants and Horticultural Plant Breeding, Department for Plant SciencesTechnical University MunichFreisingGermany
  4. 4.Unit of Fruit Science, Department for Plant SciencesTechnical University of MunichFreisingGermany
  5. 5.Biomolecular Food TechnologyTechnical University of MunichFreisingGermany

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