Tree Genetics & Genomes

, Volume 9, Issue 1, pp 75–83 | Cite as

Identification of differentially expressed genes related to coloration in red/green mutant pear (Pyrus communis L.)

  • Jun Wu
  • Guang Zhao
  • Ya-Nan Yang
  • Wen-Quan Le
  • Muhammad Awais Khan
  • Shao-Ling Zhang
  • Chao Gu
  • Wen-Jiang Huang
Original Paper


Fruit skin color is an important parameter of outer quality and plays an important role in attracting customers. In many plants, it is the result of coordinative regulation of anthocyanin pathway genes. In our study, the differential expression of cDNA library in a pair of pear mutant with red and green color was investigated to find candidate genes which might regulate the anthocyanin biosynthesis and control the coloration of pear. We constructed a cDNA library using the cDNA-amplified fragment length polymorphism approach to analyze the transcriptional differences between the original cultivar “Early red Doyenne du Comice” with high anthocyanin content in the peel and its green color mutant with comparatively low anthocyanin content. Altogether, 47 transcript-derived fragments, putatively involved in anthocyanin biosynthesis, primary metabolism, stress, and defense responses, were identified. The relationships of differentially expressed genes and coloration were investigated by quantitative real-time PCR with fruit skin samples at different developmental stages. A gene putatively involved in anthocyanin biosynthesis was found and named as PyMADS18. Its sequence is similar to genes reported in the literature as regulators of anthocyanin biosynthesis. The expression results indicate that PyMADS18 is likely to be involved in anthocyanin accumulation and regulation of anthocyanin synthesis in early fruit development of pear.


Pyrus communisPeel color Anthocyanin cDNA-AFLP PyMADS18 



cDNA-Amplified Fragment Length Polymorphism


Transcript Derived Fragments


Chalcone synthase


Flavonone 3-hydroxylase


Chalcone isomerase


Dihydroflavonol 4-reductase


Anthocyanin synthase


UDP-glucose and flavonoid 3-O-glucosyl transferase


Days After Full Blooming


Quantitative Reverse Transcription-Polymerase Chain Reaction



This study was supported by the National Science Foundation of China (30900974) and the Earmarked Fund for China Agriculture Research System (No.CARS-29-02).

Supplementary material

11295_2012_534_MOESM1_ESM.docx (279 kb)
ESM 1 (DOCX 279 kb)


  1. Allan AC, Hellens RP, Laing WA (2008) MYB transcription factors that color our fruit. Trends Plant Sci 13:99–102PubMedCrossRefGoogle Scholar
  2. Bachem CW, van der Hoeven RS, de Bruijn SM, Vreugdenhil D, Zabeau M, Visser RG (1996) Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J 9:745–753PubMedCrossRefGoogle Scholar
  3. Baisakh N, Subudhi PK, Parami NP (2006) cDNA-AFLP analysis reveals differential gene expression in response to salt stress in a halophyte Spartina alterniflora Loisel. Plant Science 170:1141–1149CrossRefGoogle Scholar
  4. Boss PK, Davies C, Robinson SP (1996) Expression of anthocyanin biosynthesis pathway genes in red and white grapes. Plant Mol Biol 32:565–569PubMedCrossRefGoogle Scholar
  5. Breyne P, Zabeau M (2001) Genome-wide expression analysis of plant cell cycle modulated genes. Current Opinion in Plant Biology 4:136–142PubMedCrossRefGoogle Scholar
  6. Breyne P, Dreesen R, Cannoot B, Rombaut D, Vandepoele K, Rombauts S, Vanderhaeghen R, InzéD ZM (2003) Quantitative cDNA-AFLP analysis for genome-wide expression studies. Mol Genet Genomics 269:173–179PubMedGoogle Scholar
  7. Busi MV, Bustamante C, D’Angelo C, Hidalgo-Cuevas M, Boggio SB, Valle EM, Zabaleta E (2003) MADS-box genes expressed during tomato seed and fruit development. Plant Mol Biol 52:801–815PubMedCrossRefGoogle Scholar
  8. Causier B, Kieffer M, Davies B (2002) MADS-box genes reach maturity. Science 296:275–276PubMedCrossRefGoogle Scholar
  9. Chen H, Guang X, Bo Z, Yang J-Y (2009) Optimization of extraction technique of anthocyanin from red peel of ‘Nanguo’ Pear. Food Science 30(08):97–100Google Scholar
  10. Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert PD (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. P Natl Acad Sci USA 93(12):6025–6030CrossRefGoogle Scholar
  11. Ditt RF, Nester EW, Comai L (2001) Plant gene expression response to Agrobacterium tumefaciens. P Natl Acad Sci USA 98:10954–10959CrossRefGoogle Scholar
  12. Dussi MC, Sugar D, Wrolstad RE (1995) Characterizing and quantifying anthocyanins in red pears and the effect of light quality on fruit color. American Society for Horticultural Science (USA) 120:785–789Google Scholar
  13. Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427PubMedCrossRefGoogle Scholar
  14. Feng S, Wang Y, Yang S, Xu Y, Chen X (2010) Anthocyanin biosynthesis in pears is regulated by a R2R3-MYB transcription factor PyMYB10. Planta 232:245–255PubMedCrossRefGoogle Scholar
  15. Fischer TC, Gosch C, Pfeiffer J, Halbwirth H, Halle C, Stich K, Forkmann G (2007) Flavonoid genes of pear (Pyrus communis). Trees 21:521–529CrossRefGoogle Scholar
  16. Fukumura R, Takahashi H, Saito T, Tsutsumi Y, Fujimori A, Sato S, Tatsumi K, Araki R, Abe M (2003) A sensitive transcriptome analysis method that can detect unknown transcripts. Nucleic Acids Res 31:94CrossRefGoogle Scholar
  17. Goff SA, Cone KC, Chandler VL (1992) Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes Dev 6:864–875PubMedCrossRefGoogle Scholar
  18. Goodrich J, Carpenter R, Coen ES (1992) A common gene regulates pigmentation pattern in diverse plant species. Cell 68:955–964PubMedCrossRefGoogle Scholar
  19. 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 Bioch 40:955–962CrossRefGoogle Scholar
  20. Hoshino A, Johzuka-Hisatomi Y, Iida S (2001) Gene duplication and mobile genetic elements in the morning glories. Gene 265:1–10PubMedCrossRefGoogle Scholar
  21. Ingram GC, Doyle S, Carpenter R, Schultz EA, Simon R, Coen ES (1997) Dual role for fimbriata in regulating floral homeotic genes and cell division in Antirrhinum. EMBO J 16(21):6521–6534PubMedCrossRefGoogle Scholar
  22. Jia HJ, Araki A, Okamoto G (2005) Influence of fruit bagging on aroma volatiles and skin coloration of ‘Hakuho’ peach (Prunus persica Batsch). Postharvest Biol Tech 35:61–68CrossRefGoogle Scholar
  23. Kano M, Takayanagi T, Harada K, Makino K, Ishikawa F (2005) Antioxidative activity of anthocyanins from purple sweet potato, Ipomoea batatas cultivar Ayamurasaki. Biosci Biotechnol Biochem 69:979–988PubMedCrossRefGoogle Scholar
  24. Kim SH, Lee JR, Hong ST, Yoo YK, An G, Kim SR (2003) Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin. Plant Science 165(2):403–413CrossRefGoogle Scholar
  25. Kipreos ET, Paqano M (2000) The F-box protein family. Genome Biol (5):30021–30027CrossRefGoogle Scholar
  26. Koornneef M (1990) Mutations affecting the testa colour in Arabidopsis. Arabidopsis Information Service 27:1–4Google Scholar
  27. Kobayashi S, Ishimaru M, Ding CK, Yakushiji H, Goto N (2001) Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Science 160:543–550PubMedCrossRefGoogle Scholar
  28. Ksenija G, Alvaro H, Schuyler SK (2004) RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Rep 22:437a–437gCrossRefGoogle Scholar
  29. Lalusin AG, Nishita K, Kim S, Ohta M, Fujimura T (2006) A new MADS-box gene (IbMADS10) from sweet potato (Ipomoea batatas (L.) Lam) is involved in the accumulation of anthocyanin. Mol Genet Genomics 275:44–54PubMedCrossRefGoogle Scholar
  30. Lancaster JE, Grant JE, Lister CE, Taylor MC (1994) Skin color in apples: influence of copigmentation and plastid pigments on shade and darkness of red color in five genotypes. J Am Soc Hortic Sci 119:63–69Google Scholar
  31. Lao M, Arencibia AD, Carmona ER, Acevedo R, Rodriguez E, LeÓn O, Santana I (2008) Differential expression analysis by cDNA-AFLP of Saccharum spp. after inoculation with the host pathogen Sporisorium scitamineum. Plant Cell Reports 27:1103–1111PubMedCrossRefGoogle Scholar
  32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods 25:402–408PubMedCrossRefGoogle Scholar
  33. Mol J, Grotewold E, Kose R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217CrossRefGoogle Scholar
  34. Nesi N, Debeaujon I, Jond C, Stewart AJ, Jenkins GI, Caboche M, Lepiniec L (2002) The transparent test 16 locus encodes the Arabidopsis Bsister MADS domain protein and is required for proper development and pigmentation of the seed coat. Plant Cell 14:2463–2479PubMedCrossRefGoogle Scholar
  35. Ni W, Xie D, Hobbie L, Feng B, Zhao D, Akkara J, Ma H (2004) Regulation of flower development in Arabidopsis by SCF complexes. Plant Physiol 134(4):1574–1585PubMedCrossRefGoogle Scholar
  36. Niu SS, Xu CJ, Zhang WS, Zhang B, Li X, Wang KL, Ferguson IB, Allan AC, Chen KS (2010) Coordinated regulation of anthocyanin biosynthesis in Chinese bayberry (Myrica rubra) fruit by a R2R3 MYB transcription factor. Planta 231:887–899PubMedCrossRefGoogle Scholar
  37. Okada K, Shimura Y (1994) Genetic analyses of signaling in flower development using Arabidopsis. Plant Mol Biol 26:1357–1377PubMedCrossRefGoogle Scholar
  38. Pierantoni L, Dondini L, Franceschi PD, Musacchi S, Winkel BSJ, Sansavini S (2010) Mapping of an anthocyanin-regulating MYB transcription factor and its expression in red and green pear, Pyrus communis. Plant Physiol Bioch 1-7Google Scholar
  39. Sassa H, Kakui H, Miyamoto M, Suzuli Y, Hanada T, Ushijima K, Kusaba M, Hirano H, Koba T (2007) S locus F-box brothers: multiple and pollen-specific F-box genes. Genetics 175:1869–1881PubMedCrossRefGoogle Scholar
  40. Schwinn K, Venail J, Shang Y, Mackay S, Alm V, Butelli E, Oyama R, Bailey P, Davies K, Martin C (2006) A small family of MYB regulatory genes controls floral pigmentation intensity and patterning in the genus Antirrhinum. Plant Cell 18:831–851PubMedCrossRefGoogle Scholar
  41. Shirley BW, Hanley S, Goodman HM (1992) Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis mutant deficient in flavonoid biosynthesis. The Plant Cell 4:333–347PubMedGoogle Scholar
  42. Shirley BW, Kubasek WL, Storz G, Bruggemann E, Koornneef M, Ausubel FM, Goodman HM (1995) Analysis of Arabidopsis mutant deficient in flavonoid biosynthesis. Plant J 8:659–671PubMedCrossRefGoogle Scholar
  43. Steyn WJ, Holcroft DM, Wand SJE, Jacobs G (2004a) Anthocyanin degradation in detached pome fruit with reference to preharvest red color loss and pigmentation patterns of blushed and fully red pears. J Am Soc Hortic Sci 129(1):13–19Google Scholar
  44. Steyn WJ, Holcroft DM, Wand SJE, Jacobs G (2004b) Regulation of pear color development in relation to activity of flavonoid enzymes. J Am Soc Hortic Sci 129(1):6–12Google Scholar
  45. Steyn WJ, Wand SJE, Holcroft DM, Jacobs G (2005) Red color development and loss in pears. Acta Horticulturae 671:79–85Google Scholar
  46. Suda I, Oki T, Masuda M, Kobayashi M, Nishiba Y, Furuta S (2003) Physiological functionality of purple-fleshed sweet potatoes containing anthocyanins and their utilization in foods. JARQ 37:167–173Google Scholar
  47. Tao B, Shu Q, Wang JJ, Zhang WB (2004) Studies on frontiers and prospects of resources of red pears and its application. Southwest China Journal Agricultural Science 47:409–412Google Scholar
  48. Theissen G, Saedler H (1995) MADS-box genes in plant ontogeny and phylogeny: Haeckel’s ‘biogenetic law’ revisited. Curr Opin Genet Dev 5:628–638PubMedCrossRefGoogle Scholar
  49. Tsuda T, Yamaguchi M, Honda C, Moriguchi T (2004) Expression of anthocyanin biosynthesis genes in the skin of peach and nectarine fruit. J Am Soc Hortic Sci 129:857–862Google Scholar
  50. Weigel D, Meyerowitz EM (1994) The ABCs of floral homeotic genes. Cell 78:203–209PubMedCrossRefGoogle Scholar
  51. West AG, Sharrocks AD (1999) MADS-box transcription factors adopt alternative mechanisms for bending DNA. Mol Biol 288:1311–1323Google Scholar
  52. Yang L, Zheng B, Mao C, Yi K, Liu F, Wu Y, Tao Q, Wu P (2003) cDNA-AFLP analysis of inducible gene expression in rice seminal root tips under a water deficit. Gene 314:141–148PubMedCrossRefGoogle Scholar
  53. Yang G, Dun L, Shu Q, Huang C (2007) Relationship of anthocyanin content, sugar content, PAL activity and Colletotrichum gloeosporioides in peel of oil tea tree. Scientia silvae sinicae 43(6):100–104Google Scholar
  54. Zhang X, Andrew CA, Yi Q, Chen L, Li K, Su Q, Su J (2011) Differential gene expression analysis of Yunnan red pear, Pyrus pyrifolia, during fruit skin coloration. Plant Mol Biol Rep 29:305–314CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jun Wu
    • 1
  • Guang Zhao
    • 1
  • Ya-Nan Yang
    • 1
  • Wen-Quan Le
    • 2
  • Muhammad Awais Khan
    • 3
  • Shao-Ling Zhang
    • 1
  • Chao Gu
    • 1
  • Wen-Jiang Huang
    • 1
  1. 1.Centre of Pear Engineering Technology ResearchNanjing Agricultural UniversityNanjingPeople’s Republic of China
  2. 2.Changli Institute of PomologyHebei Academy of Agriculture and Forestry SciencesChangliPeople’s Republic of China
  3. 3.Department of Natural Resources and Environmental SciencesUniversity of IllinoisUrbanaUSA

Personalised recommendations