, Volume 236, Issue 4, pp 1067–1080 | Cite as

Flavonoid biosynthesis-related genes in grape skin are differentially regulated by temperature and light conditions

  • Akifumi Azuma
  • Hiroshi Yakushiji
  • Yoshiko Koshita
  • Shozo Kobayashi
Original Article


Temperature and light are important environmental factors that affect flavonoid biosynthesis in grape berry skin. However, the interrelationships between temperature and light effects on flavonoid biosynthesis have not been fully elucidated at the molecular level. Here, we investigated the effects of temperature and light conditions on the biosynthesis of flavonoids (anthocyanins and flavonols) and the expression levels of related genes in an in vitro environmental experiment using detached grape berries. Sufficient anthocyanin accumulation in the grape skin was observed under a low temperature (15 °C) plus light treatment, whereas high temperature (35 °C) or dark treatment severely suppressed anthocyanin accumulation. This indicates that the accumulation of anthocyanins is dependent on both low temperature and light. qRT-PCR analysis showed that the responses of three MYB-related genes (VlMYBA1-3, VlMYBA1-2, and VlMYBA2) to temperature and light differed greatly even though the products of all three genes had the ability to regulate anthocyanin biosynthesis pathway genes. Furthermore, the expression levels of other MYB-related genes and many flavonoid biosynthesis pathway genes were regulated independently by temperature and light. We also found that temperature and light conditions affected the anthocyanin composition in the skin through the regulation of flavonoid biosynthesis pathway genes. Our results suggest that low temperature and light have a synergistic effect on the expression of genes in the flavonoid biosynthesis pathway. These findings provide new information about the relationships between environmental factors and flavonoid accumulation in grape berry skin.


Anthocyanins Flavonols Grape Skin color Light Temperature 



Abscisic acid


Anthocyanin multidrug and toxic extrusion


Chalcone isomerase


Chalcone synthase


Dihydroflavonol 4-reductase


Flavanone 3-hydroxylase


Flavonoid 3′-hydroxylase


Flavonoid 3′5′-hydroxylase


Flavonol synthase




Leucoanthocyanin dioxygenase


9-cis-epoxycarotenoid dioxygenase 1








UDP-glucose: flavonoid 3-O-glucosyltransferase



This study was supported by a grant from the research program “Elucidation of biological mechanisms of photo response and development of advanced technologies utilizing light” provided by the Ministry of Agriculture, Forestry and Fisheries of Japan.


  1. Azuma A, Kobayashi S, Mitani N, Shiraishi M, Yamada M, Ueno T, Kono A, Yakushiji H, Koshita Y (2008) Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape skin. Theor Appl Genet 117:1009–1019PubMedCrossRefGoogle Scholar
  2. Azuma A, Kobayashi S, Goto-Yamamoto N, Shiraishi M, Mitani N, Yakushiji H, Koshita Y (2009) Color recovery in berries of grape (Vitis vinifera L.) ‘Benitaka’, a bud sport of ‘Italia’, is caused by a novel allele at the VvmybA1 locus. Plant Sci 176:470–478CrossRefGoogle Scholar
  3. Azuma A, Udo Y, Sato A, Mitani N, Kono A, Ban Y, Yakushiji H, Koshita Y, Kobayashi S (2011) Haplotype composition at the color locus is a major genetic determinant of skin color variation in Vitis × labruscana grapes. Theor Appl Genet 122:1427–1438PubMedCrossRefGoogle Scholar
  4. Ban T, Ishimaru M, Kobayashi S, Shiozaki S, Goto-Yamamoto N, Horiuchi S (2003) Abscisic acid and 2,4-dichlorohenoxyacetic acid affect the expression of anthocyanin biosynthetic pathway genes in Kyoho grape berries. J Hortic Sci Biotech 78:586–589Google Scholar
  5. Baranac JM, Petranovic NA, Dimitric-Markovic JM (1997) Spectrophotometric study of anthocyan copigmentation reactions. 2. Malvin and nonglycosidized flavone quercetin. J Agric Food Chem 45:1694–1697CrossRefGoogle Scholar
  6. 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
  7. 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
  8. Boss PK, Davies C, Robinson SP (1996a) Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol Biol 32:565–569PubMedCrossRefGoogle Scholar
  9. Boss PK, Davies C, Robinson SP (1996b) Anthocyanin composition and anthocyanin pathway gene expression in grapevine sports differing in berry skin colour. Aust J Grape Wine Res 2:163–170CrossRefGoogle Scholar
  10. Bowler C, Chua NH (1994) Emerging themes of plant signal transduction. Plant Cell 16:1529–1541Google Scholar
  11. Castellarin SD, Gaspero GD (2007) Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines. BMC Plant Biol 7:46. doi: 10.1186/1471-2229-7-46 PubMedCrossRefGoogle Scholar
  12. Castellarin SD, Gaspero GD, Marconi R, Nonis A, Peterlunger E, Paillard S, Adam-Blondon AF, Testolin R (2006) Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3′-hydroxylase, flavonoid 3′,5′-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin. BMC Genom 7. doi: 10.1186/1471-2164-7-12
  13. Christie JM, Jenkins GI (1996) Distinct UV-B and UV-A/blue light signal transduction pathways induce chalcone synthase gene expression in Arabidopsis cells. Plant Cell 8:1555–1567PubMedGoogle Scholar
  14. Christie PJ, Alfenito MR, Walbot V (1994) Impact of low temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194:541–549CrossRefGoogle Scholar
  15. Coombe BG, Hale CR (1973) The hormone content of ripening grape berries and the effects of growth substance treatments. Plant Physiol 51:629–634PubMedCrossRefGoogle Scholar
  16. Cortell JM, Kennedy JA (2006) Effect of shading on accumulation of flavonoid compounds in (Vitis vinifera L.) Pinot noir fruit and extraction in a model system. J Agric Food Chem 54:8510–8520PubMedCrossRefGoogle Scholar
  17. Cutanda-Perez MC, Ageorges A, Gomez C, Vialet S, Terrier N, Romieu C, Torregrosa L (2009) Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport. Plant Mol Biol 69:633–648PubMedCrossRefGoogle Scholar
  18. Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris NN, Walker AR, Robinson SP, Bogs J (2009) The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol 151:1513–1530PubMedCrossRefGoogle Scholar
  19. Dela G, Or E, Ovadia R, Nissim-Levi A, Weiss D, Oren-Shamir M (2003) Changes in anthocyanin concentration and composition in ‘Jaguar’ rose flowers due to transient high-temperature conditions. Plant Sci 164:333–340CrossRefGoogle Scholar
  20. Deluc L, Barrieu F, Marchive C, Lauvergeat V, Decendit A, Richard T, Carde JP, Merillon JM, Hamdi S (2006) Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway. Plant Physiol 140:499–511PubMedCrossRefGoogle Scholar
  21. 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 proanthocyanin biosynthesis in developing grape berries. Plant Physiol 147:2041–2053PubMedCrossRefGoogle Scholar
  22. Downey MO, Harvey JS, Robinson SP (2003) Synthesis of flavonols and expression of flavonol synthase genes in developing grape berries of Shiraz and Chardonnay (Vitis vinifera L.). Aust J Grape Wine Res 9:110–121CrossRefGoogle Scholar
  23. Downey MO, Harvey JS, Robinson SP (2004) The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes. Aust J Grape Wine Res 10:55–73CrossRefGoogle Scholar
  24. Frohnmeyer H, Bowler C, Schafer E (1997) Evidence for some signal transduction elements involved in UV-light-dependent responses in parsley protoplasts. J Exp Bot 48:739–750CrossRefGoogle Scholar
  25. Fujita A, Goto-Yamamoto N, Aramaki I, Hashizume K (2006) Organ-specific transcription of putative flavonol synthase genes of grapevine and effects of plant hormones and shading on flavonol biosynthesis in grape berry skins. Biosci Biotechnol Biochem 70:632–638PubMedCrossRefGoogle Scholar
  26. Gambetta GA, Matthews MA, Shaghasi TH, McElrone AJ, Castellarin SD (2010) Sugar and abscisic acid signaling orthologs are activated at the onset of ripening in grape. Planta 232:219–234PubMedCrossRefGoogle Scholar
  27. Gao JJ, Shen XF, Zhang Z, Peng RH, Xiong AS, Xu J, Zhu B, Zheng JL, Yao QH (2011) The myb transcription factor MdMYB6 suppresses anthocyanin biosynthesis in transgenic Arabidopsis. Plant Cell Tissue Organ Cult 106:235–242CrossRefGoogle Scholar
  28. Harbone JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504CrossRefGoogle Scholar
  29. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083PubMedGoogle Scholar
  30. Holton TA, Brugliera F, Tanaka Y (1993) Cloning and expression of flavonol synthase from Petunia hybrida. Plant J 4:1003–1010PubMedCrossRefGoogle Scholar
  31. Jackman RL, Smith JL (1996) Anthocyanins and betalains. In: Hendry GAF, Houghton JD (eds) Natural food colorants, 2nd edn. Chapman and Hall, London, pp 244–309CrossRefGoogle Scholar
  32. Jeong ST, Goto-Yamamoto N, Kobayashi S, Esaka M (2004) Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins. Plant Sci 167:247–252CrossRefGoogle Scholar
  33. Jeong ST, Goto-Yamamoto N, Hashizume K, Esaka M (2006) Expression of the flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes and flavonoid composition in grape (Vitis vinifera). Plant Sci 170:61–69CrossRefGoogle Scholar
  34. Jeong ST, Goto-Yamamoto N, Hashizume K, Esaka M (2008) Expression of multi-copy flavonoid pathway genes coincides with anthocyanin, flavonol and flavan-3-ol accumulation of grapevine. Vitis 47:135–140Google Scholar
  35. Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, Tonelli C, Weisshaar B, Martin C (2000) Transcriptional repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis. EMBO J 19:6150–6161PubMedCrossRefGoogle Scholar
  36. Kataoka I, Kubo Y, Sugiura A, Tomana T (1984) Effects of temperature, cluster shading and some growth regulators on l-phenylalanine ammonia-lyase activity and anthocyanin accumulation on black grapes. Mem Coll Agric Kyoto Univ 124:35–44Google Scholar
  37. Kataoka I, Sugiyama A, Beppu K (2003) Role of ultraviolet radiation in accumulation of anthocyanin in berries of ‘Gros Colman’ grapes (Vitis vinifera L.). J Jpn Soc Hort Sci 72:1–6CrossRefGoogle Scholar
  38. Kliewer WM, Torres RE (1972) Effect of controlled day and night temperatures on grape coloration. Am J Enol Vitic 23:71–77Google Scholar
  39. Kobayashi S, Ishimaru M, Hiraoka K, Honda C (2002) Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis. Planta 215:924–933PubMedCrossRefGoogle Scholar
  40. Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposon-induced mutations in grape skin color. Science 304:982PubMedCrossRefGoogle Scholar
  41. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242PubMedCrossRefGoogle Scholar
  42. Kojima K, Shiozaki K, Koshita Y, Ishida M (1999) Changes of endogenous levels of ABA, IAA and GA-like substances in fruitlets of parthenocarpic persimmon. J Jpn Soc Hort Sci 68:242–247CrossRefGoogle Scholar
  43. Koshita Y, Asakura T, Fukuda H, Tsuchida Y (2007) Nighttime temperature treatment of fruit clusters of ‘Aki Queen’ grapes during maturation and its effect on the skin color and abscisic acid content. Vitis 46:208–209Google Scholar
  44. Leyva A, Jarillo JA, Salinas J, Martinez-Zapater JM (1995) Low temperature induces the accumulation of phenylalanine ammonia-lyase and chalcone synthase mRNAs of Arabidopsis thaliana in a light-dependent manner. Plant Physiol 108:39–46PubMedGoogle Scholar
  45. Lo Piero AR, Puglisi I, Rapisarda P, Petrone G (2005) Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. J Agric Food Chem 53:9083–9088PubMedCrossRefGoogle Scholar
  46. Lund ST, Peng FY, Nayar T, Reid KE, Schlosser J (2008) Gene expression analyses in individual grape (Vitis vinifera L.) berries during ripening initiation reveal that pigmentation intensity is a valid indicator of developmental staging within the cluster. Plant Mol Biol 68:301–315PubMedCrossRefGoogle Scholar
  47. Matus JT, Aquea F, Arce-Johnson P (2008) Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes. BMC Plant Biol 8:83PubMedCrossRefGoogle Scholar
  48. Matus JT, Loyola R, Vega A, Peña-Neira A, Bordeu E, Arce-Johnson P, Alcalde JA (2009) Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J Exp Bot 60:853–867PubMedCrossRefGoogle Scholar
  49. Mazza G, Fukumoto L, Delaquis P, Girard B, Ewert B (1999) Anthocyanins, phenolics, and color of Cabernet Franc, Merlot, and Pinot Noir wines from British Columbia. J Agric Food Chem 47:4009–4017PubMedCrossRefGoogle Scholar
  50. Mori K, Sugaya S, Gemma H (2005) Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition. Sci Hort 105:319–330CrossRefGoogle Scholar
  51. Mori K, Goto-Yamamoto N, Kitayama M, Hashizume K (2007) Loss of anthocyanins in red-wine grape under high temperature. J Exp Bot 58:1935–1945PubMedCrossRefGoogle Scholar
  52. Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27PubMedCrossRefGoogle Scholar
  53. Shiraishi M, Yamada M, Mitani N, Ueno T (2007) A rapid determination method for anthocyanin profiling in grape genetic resources. J Jpn Soc Hort Sci 76:28–35CrossRefGoogle Scholar
  54. Shirley BW, Hanley S, Goodman HM (1992) Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations. Plant Cell 4:333–347PubMedGoogle Scholar
  55. Shvarts M, Borochov A, Weiss D (1997) Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression. Physiol Plant 99:67–72CrossRefGoogle Scholar
  56. Sparvoli F, Martin C, Scienza A, Gavazzi G, Tonelli C (1994) Cloning and molecular analysis of structural genes involved in flavonoid and stilbene biosynthesis in grape (Vitis vinifera L.). Plant Mol Biol 24:743–755PubMedCrossRefGoogle Scholar
  57. Spayd SE, Tarara JM, Mee DL, Ferguson JC (2002) Separation of sunlight and temperature effects on the composition of Vitis vinifera cv. Merlot berries. Am J Enol Vitic 53:171–182Google Scholar
  58. Terrier N, Glissant D, Grimplet J, Barrieu F, Abbal P, Couture C, Ageorges A, Atanassova R, Leon C, Renaudin JP, Dedaldechamp F, Romieu C, Delrot S, Hamdi S (2005) Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development. Planta 222:832–847PubMedCrossRefGoogle Scholar
  59. Terrier N, Torregrosa L, Ageorges A, Vialet S, Verriès 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
  60. Tomana T, Utsunomiya N, Kataoka I (1979a) The effect of environmental temperatures on fruit ripening on the tree. I. The effect of temperatures around whole vines and clusters on the ripening of ‘Delaware’ grapes (in Japanese with English summary). Studies from Inst Hort Kyoto Univ 9:1–5Google Scholar
  61. Tomana T, Utsunomiya N, Kataoka I (1979b) The effect of environmental temperatures on fruit ripening on the tree. II. The effect of temperatures around whole vines and clusters on the coloration of ‘Kyoho’ grapes (in Japanese with english summary). J Jpn Soc Hort Sci 48:261–266CrossRefGoogle Scholar
  62. Ubi BE, Honda C, Bessho H, Kondo S, Wada M, Kobayashi S, Moriguchi T (2006) Expression analysis of anthocyanin biosynthetic genes in apple skin: effect of UV-B and temperature. Plant Sci 170:571–578CrossRefGoogle Scholar
  63. Walker AR, Lee E, Bogs J, McDavid DAJ, Thomas MR, Robinson SP (2007) White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J 49:772–785PubMedCrossRefGoogle Scholar
  64. Wheeler S, Loveys B, Ford C, Davies C (2009) The relationship between the expression of abscisic acid biosynthesis genes, accumulation of abscisic acid and the promotion of Vitis vinifera L. berry ripening by abscisic acid. Aust J Grape Wine Res 15:195–204CrossRefGoogle Scholar
  65. Winkel-Shirley B (1999) Evidence for enzyme complexes in the phenylpropanoid and flavonoid pathways. Physiol Plant 107:142–149CrossRefGoogle Scholar
  66. Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493PubMedCrossRefGoogle Scholar
  67. Yamane T, Shibayama K (2006) Effects of changes in the sensitivity to temperature on skin coloration in ‘Aki Queen’ grape berries. J Jpn Soc Hort Sci 75:458–462CrossRefGoogle Scholar
  68. Yamane T, Jeong ST, Goto-Yamamoto N, Koshita Y, Kobayashi S (2006) Effects of temperature on anthocyanin biosynthesis in grape berry skins. Am J Enol Vitic 57:54–59Google Scholar
  69. Zheng Y, Tian L, Liu H, Pan Q, Zhan J, Huang W (2009) Sugars induce anthocyanin accumulation and flavanone 3-hydroxylase expression in grape berries. Plant Growth Regul 58:251–260CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Akifumi Azuma
    • 1
  • Hiroshi Yakushiji
    • 1
  • Yoshiko Koshita
    • 2
  • Shozo Kobayashi
    • 1
  1. 1.Grape and Persimmon Research Station, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO)Higashi HiroshimaJapan
  2. 2.National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO)TsukubaJapan

Personalised recommendations