Plant Molecular Biology Reporter

, Volume 33, Issue 1, pp 1–11 | Cite as

Characterization of DNA Methylation Variations During Fruit Development and Ripening of Sweet Orange

  • Jidi Xu
  • Haidan Xu
  • Qiang Xu
  • Xiuxin Deng
Original Paper


DNA methylation is a type of epigenetic modification involved in many biological processes. Recent studies have shown that DNA methylation plays important roles in regulating fruit development and ripening. In the present study, sweet oranges at six developmental stages were investigated to understand the possible roles of DNA methylation during fruit development and ripening. Analysis of genomic DNA methylation levels in different fruit tissues and developmental stages indicated that methylation levels were not static during fruit development and ripening. To explore the possible contributions of methylation-related genes in fruit development and ripening process, the full-length coding sequences corresponding to three types of DNA methyltransferase families (CsMET1, CsCMT3, and CsDRM1), a chromatin-remodeling gene (CsDDM1), and a demethylation gene (CsDME1) were isolated. The expression profiles of these methylation-related genes were analyzed in vegetative tissues (seedlings and leaves) and reproductive tissues (three stages of flowering and six stages of fruit development). The results showed that CsDRM1 was highly expressed in seedlings, leaves, and flowers compared with other genes. During fruit development and ripening, CsMET1, CsCMT1, CsCMT2, CsDRM1, and CsDMEs showed higher expression in the peel than in the flesh. However, global DNA methylation level changes could not be explained by the expression of a single gene, suggesting that the methylation and demethylation system may cooperatively contribute to the final global DNA methylation pattern.


Methyltransferase DNA methylation Citrus sinensis Fruit ripening 



This project was supported by the Ministry of Science and Technology of China (2011CB100600) and the National Natural Science Foundation of China (31272148 and 31222047).

Supplementary material

11105_2014_732_MOESM1_ESM.docx (13.4 mb)
ESM 1 (DOCX 13753 kb)


  1. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408(6814):796CrossRefGoogle Scholar
  2. Aufsatz W, Mette MF, Matzke AJ, Matzke M (2004) The role of MET1 in RNA-directed de novo and maintenance methylation of CG dinucleotides. Plant Mol Biol 54(6):793–804PubMedCrossRefGoogle Scholar
  3. Bain JM (1958) Morphological, anatomical, and physiological changes in the developing fruit of the Valencia orange, Citrus sinensis (L) Osbeck. Aust J Bot 6(1):1–23CrossRefGoogle Scholar
  4. Bartee L, Malagnac F, Bender J (2001) Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Genes Dev 15(14):1753–1758PubMedCentralPubMedCrossRefGoogle Scholar
  5. Cao X, Jacobsen SE (2002) Role of the Arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing. Curr Biol 12(13):1138–1144PubMedCrossRefGoogle Scholar
  6. Cao H, Zhang J, Xu J, Ye J, Yun Z, Xu Q, Deng X (2012) Comprehending crystalline β-carotene accumulation by comparing engineered cell models and the natural carotenoid-rich system of citrus. J Exp Bot 63(12):4403–4417PubMedCentralPubMedCrossRefGoogle Scholar
  7. Cheng Y-J, Guo W-W, Yi H-L, Pang X-M, Deng X (2003) An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol Biol Report 21(2):177–178CrossRefGoogle Scholar
  8. Choi Y, Gehring M, Johnson L, Hannon M, Harada JJ, Goldberg RB, Jacobsen SE, Fischer RL (2002) DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Cell 110(1):33–42PubMedCrossRefGoogle Scholar
  9. Ecker JR (2013) Epigenetic trigger for tomato ripening. Nat Biotechnol 31(2):119–120PubMedCrossRefGoogle Scholar
  10. Ergle DR, Katterman FR (1961) Deoxyribonucleic acid of cotton. Plant Physiol 36(6):811PubMedCentralPubMedCrossRefGoogle Scholar
  11. Fang J, Song C, Qian J, Zhang XY, Shangguan L, Yu H, Wang X (2010) Variation of cytosine methylation in 57 sweet orange cultivars. Acta Physiol Plant 32(6):1023–1030CrossRefGoogle Scholar
  12. Fraga MF, Uriol E, Diego LB, Berdasco M, Esteller M, Canal MJ, Rodriguez R (2002) High-performance capillary electrophoretic method for the quantification of 5-methyl 2′-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues. Electrophoresis 23(11):1677–1681PubMedCrossRefGoogle Scholar
  13. Gehring M, Henikoff S (2007) DNA methylation dynamics in plant genomes. Biochim Biophys Acta 1769(5–6):276–286PubMedCrossRefGoogle Scholar
  14. Gehring M, Reik W, Henikoff S (2009) DNA demethylation by DNA repair. Trends Genet 25(2):82–90PubMedCrossRefGoogle Scholar
  15. Gong Z, Morales-Ruiz T, Ariza RR, Roldan-Arjona T, David L, Zhu JK (2002) ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111(6):803–814PubMedCrossRefGoogle Scholar
  16. Hasbún R, Valledor L, Berdasco M, Santamaría E, Canal M, Rodríguez R, Ríos D, Sánchez M (2005) In vitro proliferation and genome DNA methylation in adult chestnuts. Acta Horticult 693:333Google Scholar
  17. He G, Elling AA, Deng XW (2011) The epigenome and plant development. Annu Rev Plant Biol 62:411–435PubMedCrossRefGoogle Scholar
  18. Henikoff S, Comai L (1998) A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis. Genetics 149(1):307–318PubMedCentralPubMedGoogle Scholar
  19. Higo H, Tahir M, Takashima K, Miura A, Watanabe K, Tagiri A, Ugaki M, Ishikawa R, Eiguchi M, Kurata N, Sasaki T, Richards E, Takano M, Kishimoto N, Kakutani T, Habu Y (2012) DDM1 (decrease in DNA methylation) genes in rice (Oryza sativa). Mol Genet Genomics 287(10):785–792PubMedCrossRefGoogle Scholar
  20. Hsieh CL (2000) Dynamics of DNA methylation pattern. Curr Opin Genet Dev 10(2):224–228PubMedCrossRefGoogle Scholar
  21. Katz E, Fon M, Lee YJ, Phinney BS, Sadka A, Blumwald E (2007) The citrus fruit proteome: insights into citrus fruit metabolism. Planta 226(4):989–1005PubMedCrossRefGoogle Scholar
  22. Ladanyia M (2010) Citrus fruit: biology, technology and evaluation. San Diego, Academic Press, p 193–196Google Scholar
  23. Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11(3):204–220PubMedCentralPubMedCrossRefGoogle Scholar
  24. Li X, Zhu J, Hu F, Ge S, Ye M, Xiang H, Zhang G, Zheng X, Zhang H, Zhang S (2012) Single-base resolution maps of cultivated and wild rice methylomes and regulatory roles of DNA methylation in plant gene expression. BMC Genomics 13(1):300PubMedCentralPubMedCrossRefGoogle Scholar
  25. Lister R, O’Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, Ecker JR (2008) Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell 133(3):523–536PubMedCentralPubMedCrossRefGoogle Scholar
  26. Liu Q, Xu J, Liu Y, Zhao X, Deng X, Guo L, Gu J (2007) A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot 58(15–16):4161–4171PubMedCrossRefGoogle Scholar
  27. Manning K, Tor M, Poole M, Hong Y, Thompson AJ, King GJ, Giovannoni JJ, Seymour GB (2006) A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening. Nat Genet 38(8):948–952PubMedCrossRefGoogle Scholar
  28. Messeguer R, Ganal MW, Steffens JC, Tanksley SD (1991) Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA. Plant Mol Biol 16(5):753–770PubMedCrossRefGoogle Scholar
  29. Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marín MI, Martínez-Macías MI, Ariza RR, Roldán-Arjona T (2006) Demeter and repressor of silencing 1 encode 5-methylcytosine DNA glycosylases. Proc Natl Acad Sci U S A 103(18):6853–6858PubMedCentralPubMedCrossRefGoogle Scholar
  30. Penterman J, Zilberman D, Huh JH, Ballinger T, Henikoff S, Fischer RL (2007) DNA demethylation in the Arabidopsis genome. Proc Natl Acad Sci U S A 104(16):6752–6757PubMedCentralPubMedCrossRefGoogle Scholar
  31. Rival A, Jaligot E, Beule T, Finnegan EJ (2008) Isolation and expression analysis of genes encoding MET, CMT, and DRM methyltransferases in oil palm (Elaeis guineensis Jacq.) in relation to the ‘mantled’ somaclonal variation. J Exp Bot 59(12):3271–3281PubMedCrossRefGoogle Scholar
  32. Sato S, Tabata S, Hirakawa H, Asamizu E, Shirasawa K, Isobe S, Kaneko T, Nakamura Y, Shibata D, Egholm M (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635–641CrossRefGoogle Scholar
  33. Scharer OD, Jiricny J (2001) Recent progress in the biology, chemistry and structural biology of DNA glycosylases. Bioessays 23(3):270–281PubMedCrossRefGoogle Scholar
  34. Singh A, Zubko E, Meyer P (2008) Cooperative activity of DNA methyltransferases for maintenance of symmetrical and non-symmetrical cytosine methylation in Arabidopsis thaliana. Plant J 56(5):814–823PubMedCentralPubMedCrossRefGoogle Scholar
  35. Telias A, Lin-Wang K, 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(1):93PubMedCentralPubMedCrossRefGoogle Scholar
  36. Teyssier E, Bernacchia G, Maury S, How Kit A, Stammitti-Bert L, Rolin D, Gallusci P (2008) Tissue dependent variations of DNA methylation and endoreduplication levels during tomato fruit development and ripening. Planta 228(3):391–399PubMedCrossRefGoogle Scholar
  37. Valledor L, Hasbun R, Meijon M, Rodriguez JL, Santamaria E, Viejo M, Berdasco M, Feito I, Fraga MF, Canal MJ, Rodriguez R (2007) Involvement of DNA methylation in tree development and micropropagation. Plant Cell Tissue Org 91(2):75–86CrossRefGoogle Scholar
  38. Vining KJ, Pomraning KR, Wilhelm LJ, Priest HD, Pellegrini M, Mockler TC, Freitag M, Strauss SH (2012) Dynamic DNA cytosine methylation in the Populus trichocarpa genome: tissue-level variation and relationship to gene expression. BMC Genomics 13:27PubMedCentralPubMedCrossRefGoogle Scholar
  39. Wang Z, Meng D, Wang A, Li T, Jiang S, Cong P, Li T (2013) The methylation of the PcMYB10 promoter is associated with green-skinned sport in Max Red Bartlett pear. Plant Physiol 162(2):885–896PubMedCentralPubMedCrossRefGoogle Scholar
  40. Xu Q, Liu Y, Zhu A, Wu X, Ye J, Yu K, Guo W, Deng X (2010) Discovery and comparative profiling of microRNAs in a sweet orange red-flesh mutant and its wild type. BMC Genomics 11(1):246PubMedCentralPubMedCrossRefGoogle Scholar
  41. Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao WB, Hao BH, Lyon MP, Chen J, Gao S, Xing F, Lan H, Chang JW, Ge X, Lei Y, Hu Q, Miao Y, Wang L, Xiao S, Biswas MK, Zeng W, Guo F, Cao H, Yang X, Xu XW, Cheng YJ, Xu J, Liu JH, Luo OJ, Tang Z, Guo WW, Kuang H, Zhang HY, Roose ML, Nagarajan N, Deng XX, Ruan Y (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45(1):59–66PubMedCrossRefGoogle Scholar
  42. Yamauchi T, Moritoh S, Johzuka-Hisatomi Y, Ono A, Terada R, Nakamura I, Iida S (2008) Alternative splicing of the rice OsMET1 genes encoding maintenance DNA methyltransferase. J Plant Physiol 165(17):1774–1782PubMedCrossRefGoogle Scholar
  43. Yu K, Xu Q, Da X, Guo F, Ding Y, Deng X (2012) Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis). BMC Genomics 13:10PubMedCentralPubMedCrossRefGoogle Scholar
  44. Zemach A, Kim MY, Hsieh PH, Coleman-Derr D, Eshed-Williams L, Thao K, Harmer SL, Zilberman D (2013) The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153(1):193–205PubMedCentralPubMedCrossRefGoogle Scholar
  45. Zhang H, Zhu JK (2012) Active DNA demethylation in plants and animals. Cold Spring Harb Symp Quant Biol 77:161–173PubMedCentralPubMedCrossRefGoogle Scholar
  46. Zhong S, Fei Z, Chen YR, Zheng Y, Huang M, Vrebalov J, McQuinn R, Gapper N, Liu B, Xiang J, Shao Y, Giovannoni JJ (2013) Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening. Nat Biotechnol 31(2):154–159PubMedCrossRefGoogle Scholar
  47. Zilberman D, Cao X, Johansen LK, Xie Z, Carrington JC, Jacobsen SE (2004) Role of Arabidopsis ARGONAUTE4 in RNA-directed DNA methylation triggered by inverted repeats. Curr Biol 14(13):1214–1220PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Key Laboratory of Horticultural Plant Biology of Ministry of EducationHuazhong Agricultural UniversityWuhanPeople’s Republic of China

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