Plant Molecular Biology

, Volume 63, Issue 3, pp 307–323 | Cite as

Identification of genes associated with flesh morphogenesis during grapevine fruit development

  • Lucie Fernandez
  • Laurent Torregrosa
  • Nancy Terrier
  • Lekha Sreekantan
  • Jérôme Grimplet
  • Chris Davies
  • Mark R. Thomas
  • Charles Romieu
  • Agnès Ageorges


Fruit morphogenesis is a process unique to the angiosperms, and yet little is known about its developmental control. Following fertilization, fruits typically undergo a dramatic enlargement that is accompanied by differentiation of numerous distinct cell types. To identify genes putatively involved in the early development of grapevine fruit, we used the fleshless berry mutant (Vitis vinifera L. cv Ugni Blanc) that has dramatically reduced fruit size due to a lack of pericarp development. Using oligo-specific arrays, 53 and 50 genes were identified as being down- and up-regulated, respectively, in the mutant. In parallel, Suppression Subtractive Hybridization performed between the mutant and the wild type (WT) allowed the identification of new transcripts differentially expressed during the first stages of mutant and WT pericarp development. From this data, the picture emerged that the mutation promotes the expression of several genes related to ripening and/or to stress and impairs the expression of several regulatory genes. Among those, five genes encoding proteins previously reported to be associated with, or involved in, developmental processes in other species (a specific tissue protein 2, ATHB13, a BURP domain protein, PISTILLATA, and YABBY2), were identified and investigated further using real-time PCR and in situ hybridization. Expression in the pericarp was confirmed, specific spatial and/or temporal patterns were detected and differences were observed between the WT and the mutant during fruit development. Expression of these genes appeared to be affected during young fruit development in the mutant, suggesting that they may play a role in grape berry morphogenesis.


Development Fleshless mutant Fruit morphogenesis Transcriptome Vitis vinifera 



Suppression subtractive hybridization


In situ hybridization


Days after anthesis


Weeks post-flowering



We would like to thank Dr. Françoise Dosba and Dr. Guy Albagnac for encouraging the project and for helpful discussions, Pat Iocco for her helpful assistance at various stages of the experiments.

Supplementary material


  1. Ageorges A, Fernandez L, Vialet S, Merdinoglu D, Terrier N, Romieu C (2006) Four specific isogenes of the anthocyanin metabolic pathway are systematically co-expressed with the red colour of grape berries. Plant Sci 170:372–383CrossRefGoogle Scholar
  2. Alcantara JM, Rey PJ (2003) Conflicting selection pressures on seed size: evolutionary ecology of fruit size in a bird-dispersed tree, Olea europaea. J Evol Biol 16:1168–1176PubMedCrossRefGoogle Scholar
  3. Alvarez J, Smyth DR (2002) Crabs claw and Spatula genes regulate growth and pattern formation during gynoecium development in Arabidopsis thaliana. Int J Plant Sci 163:17–41CrossRefGoogle Scholar
  4. Bartley GE, Ishida BK (2002) Digital fruit ripening: data mining in the TIGR tomato gene index. Plant Mol Biol Rep 20:115–130Google Scholar
  5. Bartley GE, Ishida BK (2003) Developmental gene regulation during tomato fruit ripening and in-vitro sepal morphogenesis. BMC Plant Biol 3:4PubMedCrossRefGoogle Scholar
  6. Batchelor AK, Boutilier K, Miller SS, Hattori J, Bowman LA, Hu M, Lantin S, Johnson DA, Miki BLA (2002) SCB1, a BURP-domain protein gene, from developing soybean seed coats. Planta 215:523–532PubMedCrossRefGoogle Scholar
  7. 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
  8. Boss PK, Vivier M, Matsumoto S, Dry IB, Thomas MR (2001) A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development. Plant Mol Biol 45:541–553PubMedCrossRefGoogle Scholar
  9. Boutilier KA, Gines MJ, DeMoor JM, Huang B, Baszczynski CL, Iyer VN, Miki BL (1994) Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis. Plant Mol Biol 26:1711–1723PubMedCrossRefGoogle Scholar
  10. Bowman JL (2000) The YABBY gene family and abaxial cell fate. Curr Opin Plant Biol 3:17–22PubMedCrossRefGoogle Scholar
  11. Bowman JL, Smyth DR (1999) CRABS CLAW, a gene that regulates carpel and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development 126:2387–2396PubMedGoogle Scholar
  12. Cao WX, Epstein C, Liu H, DeLoughery C, Ge NX, Lin JY, Diao R, Cao H, Long F, Zhang X, Chen YD, Wright PS, Busch S, Wenck M, Wong K, Saltzman AG, Tang ZH, Liu L, Zilberstein A (2004) Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study. BMC Genomics 5:26PubMedCrossRefGoogle Scholar
  13. Chen Q, Atkinson A, Otsuga D, Christensen T, Reynolds L, Drews GN (1999) The Arabidopsis FILAMENTOUS FLOWER gene is required for flower formation. Development 126:2715–2726PubMedGoogle Scholar
  14. Cheniclet C, Rong WR, Causse M, Frangne N, Bolling L, Carde JP, Renaudin JP (2005) Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth. Plant Physiol 139:1984–1994PubMedCrossRefGoogle Scholar
  15. Chervin C, El-Kereamy A, Roustan JP, Latche A, Lamon J, Bouzayen M (2004) Ethylene seems required for the berry development and ripening in grape, a non-climacteric fruit. Plant Sci 167:1301–1305CrossRefGoogle Scholar
  16. Cleveland WS (1979) Robust locally weighted regression and smoothing scatterplots. J Am Stat Assoc 74:829–836CrossRefGoogle Scholar
  17. Cong B, Liu JP, Tanksley SD (2002) Natural alleles at a tomato fruit size quantitative trait locus differ by heterochronic regulatory mutations. Proc Natl Acad Sci USA 99:13606–13611PubMedCrossRefGoogle Scholar
  18. Coombe BG (1976) The development of fleshy fruits. Ann Rev Plant Physiol 27:507–528CrossRefGoogle Scholar
  19. Coombe BG (1995) Adoption of a system for identifying grapevine growth stages. Aust J Grape Wine Res 1:104–110Google Scholar
  20. da Silva FG, Iandolino A, Al-Kayal F, Bohlmann MC, Cushman MA, Lim H, Erqul A, Figueroa R, Kabuloglu EK, Osborne C, Rowe J, Tattersall E, Leslie A, Xu J, Baek J, Cramer GR, Cushman JC, Cook DR (2005) Characterizing the grape transcriptome. Analysis of expressed sequence tags from multiple Vitis species and development of a compendium of gene expression during berry development. Plant Physiol 139:574–597PubMedCrossRefGoogle Scholar
  21. de Folter S, Busscher J, Colombo L, Losa A, Angenent GC (2004) Transcript profiling of transcription factor genes during silique development in Arabidopsis. Plant Mol Biol 56:351–366PubMedCrossRefGoogle Scholar
  22. de Vries SC, De Vos WM, Harnsen MC, Wessels JGH (1985) A shoot-specific mRNA from pea: nucleotide sequence and regulation as compared to light-induced mRNAs. Plant Mol Biol 4:95–102CrossRefGoogle Scholar
  23. Desai S, Hill J, Trelogan L, Diatchenko L, Siebert PD (2000) Identification of differentially expressed genes by suppression subtractive hybridization. In: Hunt SP, Livesey R (eds) Functional genomics: a pratical approach. Oxford University Press, Oxford, pp81–112Google Scholar
  24. Diatchenko L, Lau YFC, 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. Proc Natl Acad Sci USA 93:6025–6030PubMedCrossRefGoogle Scholar
  25. Dinneny JR, Yanofsky MF (2005) Drawing lines and borders: how the dehiscent fruit of Arabidopsis is patterned. BioEssays 27:42–49PubMedCrossRefGoogle Scholar
  26. Drews GN, Bowman JL, Meyerowitz EM (1991) Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 65:991–1002PubMedCrossRefGoogle Scholar
  27. Eames AJ, MacDaniels LH (1947) An introduction to plant anatomy, 2nd edn. MacGraw-Hill, New York, pp427Google Scholar
  28. Emery JF, Floyd SK, Alvarez J, Eshed Y, Hawker NP, Izhaki A, Baum SF, Bowman JL (2003) Radial patterning of Arabidopsis shoots by Class III HD-ZIP and KANADI genes. Curr Biol 13:1768–1774PubMedCrossRefGoogle Scholar
  29. Eshed Y, Izhaki A, Baum SF, Floyd SK, Bowman JL (2004) Asymetric leaf development and blade expansion in Arabidopsis are mediated by KANADI and YABBY activities. Development 131:2997–3006PubMedCrossRefGoogle Scholar
  30. Fernandez L, Romieu R, Moing A, Bouquet A, Maucourt M, Thomas MR, Torregrosa L (2006a) The grapevine flb mutation: a unique genotype to investigate differences between fleshy and non-fleshy fruit. Plant Physiol 140:537–547CrossRefGoogle Scholar
  31. Fernandez L, Doligez A, Lopez G, Thomas MR, Bouquet A, Torregrosa L (2006b) Somatic chimerism, genetic inheritance and mapping of the fleshless berry (flb) mutation in grapevine (Vitis vinifera L.). Genome 49:721–728CrossRefGoogle Scholar
  32. Ferrandiz C, Pelaz S, Yanofsky MF (1999) Control of carpel and fruit development in Arabidopsis. Ann Rev Biochem 68:321–354PubMedCrossRefGoogle Scholar
  33. Fougère-Rifot M, Benharbit El, Alami N, Brun O, Bouard J (1995) Ontogenesis of the gynoecium of Vitis vinifera L. var. Chardonnay in relation to the appearance of tannic vacuoles. J Int Sci Vigne Vin 29:105–130Google Scholar
  34. Fox SA, Loh S, Thean AL, Garlepp MJ (2004) Identification of differentially expressed genes in murine mesothelioma cell lines of differing tumorigenicity using suppression subtractive hybridization. Biochim Biophys Acta 1688:237–244PubMedGoogle Scholar
  35. Frary A, Nesbitt TC, Grandillo S, van der Knaap E, Cong B, Liu JP, Meller J, Elber R, Alpert KB, Tanksley SD (2000) fw2.2: a quantitative trait locus key to the evolution of tomato fruit size. Science 289:85–88PubMedCrossRefGoogle Scholar
  36. Giovannoni JJ (2004) Genetic regulation of fruit development and ripening. Plant Cell 16:S170–S180PubMedCrossRefGoogle Scholar
  37. Golz JF, Roccaro M, Kuzoff R, Hudson A (2004) GRAMINIFOLIA promotes growth and polarity of Antirrhinum leaves. Development 131:3661–3670PubMedCrossRefGoogle Scholar
  38. Goto K, Meyerowitz EM (1994) Function and Regulation of the Arabidopsis floral homeotic gene PISTILLATA. Genes Dev 8:1548–1560PubMedCrossRefGoogle Scholar
  39. Granger C, Coryell V, Khanna A, Keim P, Vodkin L, Shoemaker RC (2002) Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean. Genome 45:693–701PubMedCrossRefGoogle Scholar
  40. Grimplet J, Romieu C, Audergon JM, Albagnac G, Lambert P, Bouchet JP, Marty I, Terrier N (2005) Transcriptomic study of apricot fruit (Prunus armeniaca) ripening amongst 13006 EST. Physiol Plant 125:281–292CrossRefGoogle Scholar
  41. Gu Q, Ferrandiz C, Yanofsky MF, Martienssen R (1998) The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 125:1509–1517PubMedGoogle Scholar
  42. Hanson J, Johannesson H, Engstrom P (2001) Sugar-dependent alterations in cotyledon and leaf development in transgenic plants expressing the HDZhdip gene ATHB13. Plant Mol Biol 45:247–262PubMedCrossRefGoogle Scholar
  43. Hanson J, Regan S, Engstrom P (2002) The expression pattern of the homeobox gene ATHB13 reveals a conservation of transcriptional regulatory mechanisms between Arabidopsis and hybrid aspen. Plant Cell Rep 21:81–89CrossRefGoogle Scholar
  44. Hardie WJ, O’Brien TP, Jaudzems VG (1996) Morphology, anatomy and development of the pericarp after anthesis in grape, Vitis vinifera L. Aust J Grape Wine Res 2:97–142Google Scholar
  45. Hattori J, Boutilier KA, Campagne MMV, Miki BL (1998) A conserved BURP domain defines a novel group of plant proteins with unusual primary structures. Mol Gen Genet 259:424–428PubMedCrossRefGoogle Scholar
  46. Huang XQ, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877PubMedCrossRefGoogle Scholar
  47. Jackson D (1991) In situ hybridization in plants. In: Gurr SJ, McPherson M, Bowles DJ (eds) Molecular plant pathology, a practical approach, vol 1. Oxford University Press, Oxford, pp163–174Google Scholar
  48. Jang S, Hur J, Kim SJ, Han MJ, Kim SR, An G (2004) Ectopic expression of OsYAB1 causes extra stamens and carpels in rice. Plant Mol Biol 56:133–143PubMedCrossRefGoogle Scholar
  49. Knapp S (2002) Tobacco to tomatoes: a phylogenetic perspective on fruit diversity in the Solanaceae. J Exp Bot 53:2001–2022PubMedCrossRefGoogle Scholar
  50. Kumaran MK, Bowman JL, Sundaresan V (2002) YABBY polarity genes mediate the repression of KNOX homeobox genes in Arabidopsis. Plant Cell 14:2761–2770PubMedCrossRefGoogle Scholar
  51. Lemaire-Chamley M, Petit J, Garcia V, Just D, Baldet P, Germain V, Fagard M, Mouassite M, Cheniclet C, Rothan C (2005) Changes in transcriptional profiles are associated with early fruit tissue specialization in tomato. Plant Physiol 139:750–769PubMedCrossRefGoogle Scholar
  52. Linke B, Nothnagel T, Borner T (2003) Flower development in carrot CMS plants: mitochondria affect the expression of MADS box genes homologous to GLOBOSA and DEFICIENS. Plant J 34:27–37PubMedCrossRefGoogle Scholar
  53. Liu JP, Van Eck J, Cong B, Tanksley SD (2002) A new class of regulatory genes underlying the cause of pear-shaped tomato fruit. Proc Natl Acad Sci USA 99:13302–13306PubMedCrossRefGoogle Scholar
  54. Munoz FJ, Dopico B, Labrador E (1997) Two growth-related organ-specific cDNAs from Cicer arietinum epicotyls. Plant Mol Biol 35:433–442PubMedCrossRefGoogle Scholar
  55. Navarro C, Efremova N, Golz JF, Rubiera R, Kuckenberg M, Castillo R, Tietz O, Saedler H, Schwarz-Sommer Z (2004) Molecular and genetic interactions between STYLOSA and GRAMINIFOLIA in the control of Antirrhinum vegetative and reproductive development. Development 131:3649–3659PubMedCrossRefGoogle Scholar
  56. Ojeda H, Deloire A, Carbonneau A, Ageorges A, Romieu C (1999) Berry development of grapevines: relations between the growth of berries and their DNA content indicate cell multiplication and enlargement. Vitis 38:145–150Google Scholar
  57. Ranjan P, Kao YY, Jiang HY, Joshi CP, Harding SA, Tsai CJ (2004) Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism. Planta 219:694–704PubMedCrossRefGoogle Scholar
  58. Rezaian MA, Krake LR (1987) Nucleic acid extraction and virus detection in grapevine. J Virol Methods 17:277–285PubMedCrossRefGoogle Scholar
  59. Sawa S, Ito T, Shimura Y, Okada K (1999) FILAMENTOUS FLOWER controls the formation and development of Arabidopsis inflorescences and floral meristems. Plant Cell 11:69–86PubMedCrossRefGoogle Scholar
  60. Sessa G, Carabelli M, Ruberti I, Lucchetti S, Baima S, Morelli G (1994) Identification of distinct families of HD-Zip proteins in Arabidopsis thaliana. In: Puigdoménech P, Coruzzi G (eds) Analysis of plant development and metabolism. Springer-Verlag, Berlin/Heidelberg, pp411–426Google Scholar
  61. Siegfried KR, Eshed Y, Baum SF, Otsuga D, Drews GN, Bowman JL (1999) Members of the YABBY gene family specify abaxial cell fate in Arabidopsis. Development 126:4117–4128PubMedGoogle Scholar
  62. Sreekantan L, Torregrosa L, Fernandez L, Thomas MR (2006) VvMADS9, a class B MADS-box gene involved in grapevine flowering, shows different expression patterns in mutants with abnormal petal and stamen structures. Funct Plant Biol 33:877–886CrossRefGoogle Scholar
  63. Terrier N, Glissant D, Grimplet J, Barrieu F, Abbal P, Couture C, Ageorges A, Atanassova R, Léon C, Renaudin JP, Dedaldéchamp 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
  64. Tesniere C, Vayda ME (1991) Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Mol Biol Rep 9:242–251Google Scholar
  65. Theissen G (2001) Development of floral organ identity: stories from the MADS house. Curr Opin Plant Biol 4:75–85PubMedCrossRefGoogle Scholar
  66. Wang AM, Xia Q, Xie WS, Datla R, Selvaraj G (2003) The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development. Proc Natl Acad Sci USA 100:14487–14492PubMedCrossRefGoogle Scholar
  67. Waters DLE, Holton TA, Ablett EM, Lee LS, Henry RJ (2005) cDNA microarray analysis of developing grape (Vitis vinifera cv. Shiraz) berry skin. Funct Integr Genomics 5:40–58PubMedCrossRefGoogle Scholar
  68. Williams ME, Mundy J, Kay SA, Chua NH (1990) Differential expression of two related organ-specific genes in pea. Plant Mol Biol 14:765–774PubMedCrossRefGoogle Scholar
  69. Yamada T, Ito M, Kato M (2004) YABBY2-homologue expression in lateral organs of Amborella trichopoda (Amborellaceae). Int J Plant Sci 165:917–924CrossRefGoogle Scholar
  70. Yamaguchi T, Nagasawa N, Kawasaki S, Matsuoka M, Nagato Y, Hirano HY (2004) The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa. Plant Cell 16:500–509PubMedCrossRefGoogle Scholar
  71. Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple systematic variation. Nucleic Acid Res 30:e15PubMedCrossRefGoogle Scholar
  72. Yao JL, Dong YH, Morris BAM (2001) Parthenocarpic apple fruit production conferred by transposon insertion mutations in a MADS-box transcription factor. Proc Natl Acad Sci USA 98:1306–1311PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Lucie Fernandez
    • 1
  • Laurent Torregrosa
    • 1
  • Nancy Terrier
    • 2
  • Lekha Sreekantan
    • 3
  • Jérôme Grimplet
    • 2
  • Chris Davies
    • 3
  • Mark R. Thomas
    • 3
  • Charles Romieu
    • 1
  • Agnès Ageorges
    • 2
  1. 1.UMR BEPCMontpellier, Cedex 01France
  2. 2.UMR SPOMontpellier, Cedex 01France
  3. 3.CSIRO Plant IndustryGlen OsmondAustralia

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