, Volume 230, Issue 5, pp 899–915 | Cite as

Expression balances of structural genes in shikimate and flavonoid biosynthesis cause a difference in proanthocyanidin accumulation in persimmon (Diospyros kaki Thunb.) fruit

  • Takashi Akagi
  • Ayako Ikegami
  • Yasuhiko Suzuki
  • Junya Yoshida
  • Masahiko Yamada
  • Akihiko Sato
  • Keizo YonemoriEmail author
Original Article


Persimmon fruits accumulate a large amount of proanthocyanidin (PA) during development. Fruits of pollination-constant and non-astringent (PCNA) type mutants lose their ability to produce PA at an early stage of fruit development, while fruits of the normal (non-PCNA) type remain rich in PA until fully ripened. To understand the molecular mechanism for this difference, we isolated the genes involved in PA accumulation that are differentially expressed between PCNA and non-PCNA, and confirmed their correlation with PA content and composition. The expression of structural genes of the shikimate and flavonoid biosynthetic pathways and genes encoding transferases homologous to those involved in the accumulation of phenolic compounds were downregulated coincidentally only in the PCNA type. Analysis of PA composition using the phloroglucinol method suggested that the amounts of epigallocatechin and its 3-O-gallate form were remarkably low in the PCNA type. In the PCNA type, the genes encoding flavonoid 3′5′ hydroxylase (F3′5′H) and anthocyanidin reductase (ANR) for epigallocatechin biosynthesis showed remarkable downregulation, despite the continuous expression level of their competitive genes, flavonoid 3′ hydroxylation (F3′H) and leucoanthocyanidin reductase (LAR). We also confirmed that the relative expression levels of F3′5H to F3H, and ANR to LAR, were considerably higher, and the PA composition corresponded to the seasonal expression balances in both types. These results suggest that expressions of F35H and ANR are important for PA accumulation in persimmon fruit. Lastly, we tested enzymatic activity of recombinant DkANR in vitro, which is thought to be an important enzyme for PA accumulation in persimmon fruits.


Diospyros Flavonoid Polymerisation Proanthocyanidin Shikimate pathway 



4-Coumarate: coenzyme A ligase


Anthocyanidin reductase


Anthocyanidin synthase






Chalcone isomerase


Chalcone synthase


Chorismate synthase


3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase


Dihydroflavonol 4-reductase


3-Dehydroquinate dehydratase/shikimate 5-dehydrogenase


3-Dehydroquinate synthase






Epigallocatechin gallate


5-Enolpyruvylshikimate-3-phosphte synthase


Flavonoid 3-O-galactosyltransferase


Flavanone 3-hydroxylase


Flavanone 3′-hydroxylase


Flavanone 3′5′-hydroxylase




Glutathione S-transferase


Leucoanthocyanidin reductase


Phenylalanine ammonia lyase




Pollination-constant and non-astringent mutants


Serine carboxypeptidase like


Shikimate kinase


Suppression subtractive hybridisation



We are grateful to Dr. Shozo Kobayashi (Department of Grape and Persimmon Research, National Institute of Fruit Tree Science, Japan) for helpful discussion, and Dr. Kentaro Inoue (Department of Plant Sciences, University of California, Davis, USA) for critical advices and providing the HPLC equipments, and Dr. Richard A. Dixon (Plant Biology Division, Samuel Roberts Noble Foundation, USA) for providing pMMtANR, which encodes N-terminal in-frame fusion of MtANR with a maltose-binding protein.

Supplementary material

425_2009_991_MOESM1_ESM.doc (148 kb)
Supplementary material (DOC 148 kb)


  1. Abrahams S, Tanner GJ, Larkin PJ, Ashton AR (2002) Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis. Plant Physiol 130:561–576PubMedCrossRefGoogle Scholar
  2. Aron PM, Kennedy JA (2008) Flavan-3-ols: nature, occurrence and biological activity. Mol Nutr Food Res 52:79–104PubMedCrossRefGoogle Scholar
  3. Bogs J, Downey MO, Harvey JS, Ashton AR, Tanner GJ, Robinson SP (2005) Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves. Plant Physiol 139:652–663PubMedCrossRefGoogle Scholar
  4. Bogs J, Ebadi A, McDavid D, Robinson SP (2006) Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. Plant Physiol 140:279–291PubMedCrossRefGoogle Scholar
  5. Bogs J, Jaffé FW, Takos AM, Walker AR, Robinson SP (2007) The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol 143:1347–1361PubMedCrossRefGoogle Scholar
  6. Brugliera F, Barri-Rewell G, Holton TA, Mason JG (1999) Isolation and characterization of a flavonoid 3′-hydroxylase cDNA clone corresponding to the Ht1 locus of Petunia hybrida. Plant J 19:441–451PubMedCrossRefGoogle Scholar
  7. Castellarin SD, Matthews MA, Di Gaspero G, Gambetta GA (2007) Water deficit accelerate ripening and induce change in gene expression regulating flavonoid biosynthesis in grape berries. Planta 227:10–112CrossRefGoogle Scholar
  8. Cos P, De Bruyne T, Hermans N, Apers S, Berghe DV, Vlietinck AJ (2004) Proanthocyanidins in health care: current and new trends. Curr Med Chem 11:1345–1359PubMedGoogle Scholar
  9. de Vetten N, ter Horst J, van Schaik HP, de Boer A, Mol J, Koes R (1999) A cytochrome b(5) is required for full activity of flavonoid 3′, 5′-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci USA 96:778–783PubMedCrossRefGoogle Scholar
  10. Deluc L, Bogs J, Walker AR, Ferrier T, Decendit A, Merillon JM, Robinson SP, Barrieu F (2008) The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries. Plant Physiol 147:2041–2053PubMedCrossRefGoogle Scholar
  11. Ding L, Hofius D, Hajirezaei MR, Fernie AR, Bornke F, Sonnewald U (2007) Functional analysis of the essential bifunctional tobacco enzyme 3-dehydroquinate dehydratase/shikimate dehydrogenase in transgenic tobacco plants. J Exp Bot 58:2053–2067PubMedCrossRefGoogle Scholar
  12. Dixon DP, Lapthorn A, Edwards R (2002) Plant glutathione transferases. Genome Biol 3:S3004.1–S300410Google Scholar
  13. Dixon RA, Xie D-Y, Sharma SB (2005) Proanthocyanidins—a final frontier in flavonoid research? New Phytol 165:9–28PubMedCrossRefGoogle Scholar
  14. Downey MO, Harvey JS, Robinson SP (2003) Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. Aust J Grape Wine Res 9:15–27CrossRefGoogle Scholar
  15. Gross G (1999) Biosynthesis of hydrolysable tannins. In: Pino B (ed) Comprehensive natural products chemistry. Carbohydrates and their derivatives including tannins, cellulose and related lignins, vol 3. Elsevier, Amsterdam, pp 799–826Google Scholar
  16. Gu H, Li C, Xu Y, Hu W, Chen M (2008) Structural features and antioxidant activity of tannin from persimmon pulp. Food Res Int 41:208–217CrossRefGoogle Scholar
  17. Guan KL, Dixon JE (1991) Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem 192:262–267PubMedCrossRefGoogle Scholar
  18. Herrmann KM (1995) The shikimate pathway as an entry to aromatic secondary metabolism. Plant Physiol 107:7–12PubMedCrossRefGoogle Scholar
  19. Herrmann KM, Weaver LM (1999) The shikimate pathway. Annu Rev Plant Physiol Plant Mol Biol 50:473–503PubMedCrossRefGoogle Scholar
  20. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083PubMedCrossRefGoogle Scholar
  21. Holton TA, Brugliera F, Lester DR, Tanaka Y, Hyland GD, Menting JGT, Lu CY, Farcy E, Stevenson TW, Cornish EC (1993) Cloning and expreesion of cytochrome-P450 gene-controlling flower color. Nature 366:276–279PubMedCrossRefGoogle Scholar
  22. Ikegami A, Kitajima A, Yonemori K (2005a) Inhibition of flavonoid biosynthetic gene expression coincides with loss of astringency in pollination-constant, non-astringent (PCNA)-type persimmon fruit. J Hortic Sci Biotechnol 80:225–228Google Scholar
  23. Ikegami A, Sato A, Yamada M, Kitajima A, Yonemori K (2005b) Molecular size profiles of tannins in persimmon fruits of Japanese and Chinese pollination-constant non-astringent (PCNA)-type cultivars and their offspring revealed by size-exclusion chromatography. J Jpn Soc Hortic Sci 74:437–443CrossRefGoogle Scholar
  24. Ikegami A, Sato A, Yamada M, Kitajima A, Yonemori K (2005c) Expression of genes involved in proanthocyanidin biosynthesis during fruit development in a Chinese pollination-constant, nonastringent (PCNA) persimmon, ‘Luo Tian Tian Shi’. J Am Soc Hortic Sci 130:830–835Google Scholar
  25. Ikegami A, Eguchi S, Sato A, Yamada M, Kitajima A, Mitani N, Yonemori K (2006) Segregations of astringent progenies in the F1 populations derived from crosses between a Chinese pollination-constant non-astringent (PCNA) ‘Luo Tian Tian Shi’, and Japanese PCNA and pollination-constant, astringent (PCA) cultivars. HortScience 41:561–563Google Scholar
  26. Ikegami A, Eguchi S, Kitajima A, Inoue K, Yonemori K (2007) Identification of genes involved in proanthocyanidin biosynthesis of persimmon (Diospyros kaki) fruit. Plant Sci 172:1037–1047CrossRefGoogle Scholar
  27. Ikegami A, Akagi T, Potter D, Yamada M, Sato A, Yonemori K, Kitajima A, Inoue K (2009) Molecular identification of 1-cys peroxiredoxin and anthocyanidin/flavonol 3-O-galactosyltransferase from proanthocyanidin-rich young fruits of persimmon (Diospyros kaki Thunb.). Planta. doi: 10.1007/s00425-009-0989-0 Google Scholar
  28. Jones JD, Henstrand JM, Handa AK, Herrmann KM, Weller SC (1995) Impaired wound induction of 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase and altered stem development in transgenic potato plants expressing a DAHP synthase antisense construct. Plant Physiol 108:1413–1421PubMedGoogle Scholar
  29. Kanzaki S, Sato A, Yamada M, Yonemori K, Sugiura A (2001) Identification of molecular markers linked to the trait of natural astringency loss Japanese persimmon (Diospyros kaki) fruit. J Am Soc Hortic Sci 126:51–55Google Scholar
  30. Kennedy JA, Jones GP (2001) Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J Agric Food Chem 49:1740–1746PubMedCrossRefGoogle Scholar
  31. Lepiniec L, Debeaujon I, Routaboul JM, Baudry A, Pourcel L, Nesi N, Caboche M (2006) Genetics and biochemistry of seed flavonids. Annu Rev Plant Biol 57:405–430PubMedCrossRefGoogle Scholar
  32. Letham DS (1960) The separation of plant cells with ethylenediamineteraacetic acid. Exp Cell Res 21:353–360PubMedCrossRefGoogle Scholar
  33. Lillo C, Lea US, Ruoff P (2007) Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway. Plant Cell Environ 31:587–601PubMedCrossRefGoogle Scholar
  34. Matsuo T, Itoo S (1978) The chemical structure of kaki-tannin from immature fruit of the persimmon (Diospyros kaki L.). Agric Biol Chem 42:1637–1643CrossRefGoogle Scholar
  35. McMahon LR, McAllister TA, Berg BP, Majak W, Acharya SN, Popp JD, Coulman BE, Wang Y, Cheng KJ (2000) A review of the effects of forage condensed tannins on ruminal fermentation and bloat in grazing cattle. Can J Plant Sci 80:469–485CrossRefGoogle Scholar
  36. Milkowski C, Strack D (2004) Serine carboxypeptidase-like acyltransferase. Phytochemistry 65:517–524PubMedCrossRefGoogle Scholar
  37. Nesi N, Jond C, Debeaujon I, Caboche M, Lepiniec L (2001) The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell 13:2099–2114PubMedCrossRefGoogle Scholar
  38. Oshida MK, Yonemori K, Sugiura A (1996) On the nature of coagulated tannins in astringent-type persimmon fruit after an artificial treatment of astringency removal. Postharvest Biol Technol 8:317–327CrossRefGoogle Scholar
  39. Pang Y, Peel GJ, Wright E, Wang Z, Dixon RA (2007) Early steps in proanthocyanidin biosynthesis in the model legume Medicago truncatula. Plant Physiol 145:601–615PubMedCrossRefGoogle Scholar
  40. Pang Y, Peel GJ, Sharma SB, Tang Y, Dixon RA (2008) A transcript profiling approach reveals an epicatechin-specific glucosyltransferase expressed in the seed coat of Medicago truncatula. Proc Natl Acad Sci USA 105:14210–14215PubMedCrossRefGoogle Scholar
  41. Paolocci F, Robbins MP, Madeo L, Arcioni S, Martens S, Damiani F (2007) Ectopic expression of Basic Helix-Loop-Helix gene transactivates parallel pathways of proanthocyanidin biosynthesis. Structure, Expression analysis, and genetic control of leucoanthocyanidin 4-reductase and anthocyanidin reductase gene in Lotus corniculatus. Plant Physiol 143:504–516PubMedCrossRefGoogle Scholar
  42. Petrucco S, Bolchi A, Foroni C, Percudani R, Rosi GL, Ottonello S (1996) A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductase is activated in response to sulfur starvation. Plant Cell 8:69–80PubMedCrossRefGoogle Scholar
  43. Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11:1433–1444PubMedCrossRefGoogle Scholar
  44. Rebrikov DV, Britanova OV, Gurskaya NG, Lukyanov KA, Tarabykin VS, Lukyanov SA (2000) Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization. Nucleic Acids Res 28(20):e90Google Scholar
  45. Sarma AD, Sharma R (1999) Purification and characterization of UV-B induced phenylalanine ammonia-lyase from rice seedlings. Phytochemistry 49:729–737CrossRefGoogle Scholar
  46. Suzuki T, Someya S, Hu F, Tanokura M (2005) Comparative study of catechin compositions in five Japanese persimmons (Diospyros kaki). Food Chem 93:149–152CrossRefGoogle Scholar
  47. Suzuki Y, Kamitakahara H, Takano T, Nakatsubo F, Yonemori K (2009) Identification of condensed tannins in kaki (Diospyros kaki) fruit and their constitutive differences among cultivars. Hortic Res (Japan) 8(supple 1):72Google Scholar
  48. Taira S (1996) Astringency in persimmon. In: Linskens HF, Jackson JF (eds) Fruit analysis. Springer, Berlin, pp 97–110Google Scholar
  49. Takos AM, Jaffe 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 142:1216–1232PubMedCrossRefGoogle Scholar
  50. Tanner GJ, Francki KT, Abrahams S, Watson JM, Larkin PJ, Ashton AR (2003) Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA. J Biol Chem 278:31647–31656PubMedCrossRefGoogle Scholar
  51. Terrier T, Torregrosa L, Ageorges A, Vialet S, Verries C, Cheynier V, Romieu C (2009) Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol 149:1028–1041PubMedCrossRefGoogle Scholar
  52. Tohge T, Nishiyama Y, Hirai MY, Yano M, Nakajima J, Awazuhara M, Inoue E, Takahashi H, Goodenowe DB, Kitayama M, Noji M, Yamazaki M, Saito K (2005) Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor. Plant J 42:218–235PubMedCrossRefGoogle Scholar
  53. Ushijima K, Sassa H, Dandekar AM, Gradziel TM, Tao R, Hirano H (2003) Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism. Plant Cell 15:771–781PubMedCrossRefGoogle Scholar
  54. Wan CY, Wilkins TA (1994) A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem 223:7–12PubMedCrossRefGoogle Scholar
  55. Werner RA, Bacher A, Eisenrich W (1999) Analysis of gallic acid biosynthesis via quantitative prediction of isotope labeling patterns. In: Gross G, Hemingway R, Yoshida T (eds) Plant polyphenols, chemistry, biology, pharmacology, vol 2. Kluwer /Plenum Publisher, Dordrecht, pp 43–61Google Scholar
  56. Werner RA, Rossmann A, Schwarz C, Bacher A, Schmidt H-L, Eisenreich W (2004) Biosynthesis of gallic acid in Rhus typhina: discrimination between alternative pathways from natural oxygen isotope abundance. Phytochemistry 65:2809–2813PubMedCrossRefGoogle Scholar
  57. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493PubMedCrossRefGoogle Scholar
  58. Xie D-Y, Dixon RA (2005) Proanthocyanidin biosynthesis—still more questions than answers? Phytochemistry 66:2127–2144PubMedCrossRefGoogle Scholar
  59. Xie D-Y, Sharma SB, Paiva NL, Ferreira D, Dixon RA (2003) Role of anthocyanidin reductase, encoded by BANYULS in plant flavonoid biosynthesis. Science 299:396–399PubMedCrossRefGoogle Scholar
  60. Xie D-Y, Sharma SB, Dixon RA (2004) Anthocyanidin reductases from Medicago truncatula and Arabidopsis thaliana. Arch Biochem Biophys 422:91–102PubMedCrossRefGoogle Scholar
  61. Yonemori K, Matsushima J (1985) Property of development of the tannin cells from non-astringent and astringent type fruits of Japanese persimmon (Diospyros kaki) and its relationship to natural astringency. J Jpn Soc Hortic Sci 54:201–208 (in Japanese with English summary)CrossRefGoogle Scholar
  62. Yonemori K, Matsushima J, Sugiura A (1983) Differences in tannin of non-astringent and astringent type fruits of Japanese persimmon (Diospyros kaki Thunb). J Jpn Soc Hortic Sci 52:135–144 (in Japanese with English summary)CrossRefGoogle Scholar
  63. Yonemori K, Sugiura A, Yamada M (2000) Persimmon genetics and breeding. In: Janick J (ed) Plant breeding reviews, 19th edn. Wiley, New York, pp 191–225Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Takashi Akagi
    • 1
  • Ayako Ikegami
    • 2
  • Yasuhiko Suzuki
    • 1
  • Junya Yoshida
    • 3
  • Masahiko Yamada
    • 4
  • Akihiko Sato
    • 5
  • Keizo Yonemori
    • 1
    Email author
  1. 1.Laboratory of Pomology, Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Laboratory of Pomology, Department of Bioproduction SciencesIshikawa Prefectural UniversityNonoichiJapan
  3. 3.Agricultural Products Distribution DivisionTakamatsuJapan
  4. 4.Department of Citrus ResearchNational Institute of Fruit Tree ScienceKuchinotsuJapan
  5. 5.Grape and Persimmon Research StationNational Institute of Fruit Tree ScienceAkitsu, Higashi-HiroshimaJapan

Personalised recommendations