Molecular Biology Reports

, Volume 37, Issue 8, pp 3851–3862 | Cite as

Molecular analysis and expression of a floral organ-specific polygalacturonase gene isolated from rapeseed (Brassica napus L.)

  • Lili Wan
  • Xiuyun Xia
  • Dengfeng Hong
  • Guangsheng Yang


High throughput screening of stage-specific differentially expressed genes in a Brassica napus two-line Rs1046A/B subtractive library was used to identify the BnQRT3 gene associated with cell wall metabolism. Phylogenetic analysis indicates the protein product of BnQRT3 is polygalacturonase. According to cytological comparisons of Rs1046 sterile and fertile anthers, RT–PCR studies and in situ hybridizations, BnQRT3 is expressed most strongly in floral organs and may play an essential role in pollen maturation. Analysis of the histological staining pattern of BnQRT3 promoter-GUS constructs in transgenic Arabidopsis and Brassica napus revealed that proximal part of 5′-flanking region directed expression in the vascular tissue of filaments, veins in sepal and petals, stigma, branch connective and the floral organ abscission zone during the open flower stage. In the meanwhile, Activity of BnQRT3 was detected in the anthers, which commences at the microsporocyte stage and persists as anther approaches dehiscence. Strong GUS expression also can be observed in the vascular tissue of leaves and stem by compression with forceps or excision, suggesting that the BnQRT3 promoter is responsive to wounding.


Floral organ BnQRT3 Promoter Vascular tissue Wounding 

Supplementary material

11033_2010_41_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 32 kb)
11033_2010_41_MOESM2_ESM.doc (711 kb)
Supplementary material 2 (DOC 711 kb)
11033_2010_41_MOESM3_ESM.doc (388 kb)
Supplementary material 3 (DOC 388 kb)


  1. 1.
    Yang CY, Vizcay-Barrena G, Conner K, Wilson ZA (2007) Male sterility1 is required for tapetal development and pollen wall biosunthesis. Plant Cell 19:3530–3548CrossRefPubMedGoogle Scholar
  2. 2.
    Scott RJ, Spielman M, Dickinson HG (2004) Stamen structure and function. Plant Cell 16(Suppl 1):S46–S60CrossRefPubMedGoogle Scholar
  3. 3.
    Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu Y-C, Lee PY, Truong MT, Beals TP, Goldberg RB (1999) Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11:297–322CrossRefGoogle Scholar
  4. 4.
    Ma H (2005) Molecular gentic analysis of microsporogenesis and microgametogenesis in flowering plants. Ann Rev Plant Biol 56:393–434CrossRefGoogle Scholar
  5. 5.
    Deng W, Zhou L, Zhou YT, Wang YJ, Wang ML, Zhao Y (2010) Isolation and characterization of three duplicated PISTILLATA genes in Brassica napus. Mol Biol Rep DOI 10.1007/s11033-010-9981-9
  6. 6.
    Parkin IAP, Sharpe AG, Lydiate DJ (2003) Patterns of genome duplication within the Brassica napus genome. Genome 46:291–303CrossRefPubMedGoogle Scholar
  7. 7.
    Huang L, Cao JS, Zhang AH, Ye YQ (2008) Characterization of a putative pollen-specific arabinogalactan protein gene, BcMF8, from Brassica campestris ssp. Chinensis. Mol Biol Rep 35:631–639CrossRefGoogle Scholar
  8. 8.
    Song JH, Zhang LX, Cao JS (2009) Molecular cloning and characterization of a novel pollen predominantly membrane protein gene BcMF12 from Brassica campestris ssp. Chinensis. Mol Biol Rep 36:2307–2314CrossRefGoogle Scholar
  9. 9.
    Li YY, Cao JS (2009) Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis. Mol Biol Rep 36:929–937CrossRefPubMedGoogle Scholar
  10. 10.
    Carlsson J, Lagercrantz U, Sundstrom J, Teixeira R, Wellmer F, Meyerowitz EM, Glimelius K (2007) Microarray analysis reveals altered expression of a large number of nuclear genes in developing cytoplasmic male sterile Brassica napus flowers. Plant J 49:452–462CrossRefPubMedGoogle Scholar
  11. 11.
    Kang J, Zhang G, Bonnema G, Fang Z, Wang X (2008) Global analysis of gene expression in flower buds of Ms-cd1 Brassica oleracea conferring male sterility by using an Arabidopsis microarray. Plant Mol Biol 66:177–192CrossRefPubMedGoogle Scholar
  12. 12.
    Wu JY, Shen JR, Mao XZ, Liu KD, Wei LP, Liu PW, Yang GS (2007) Isolation and analysis of differentially expressed genes in dominant genic male sterility (DGMS) Brassica napus L. using subtractive PCR and cDNA microarray. Plant Sci 172:204–211CrossRefGoogle Scholar
  13. 13.
    Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinformatics 5:150–163CrossRefPubMedGoogle Scholar
  14. 14.
    Kouchi H, Hata S (1993) Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Mol Gen Genet 238:106–119PubMedGoogle Scholar
  15. 15.
    Mizuno S, Osakabe Y, Maruyama K, Ito T, Osakabe K, Sato T, Shinozaki K, Yamaguchi-Shinozaki K (2007) Receptor-like protein kinase 2 (RPK2) is a novel factor controlling anther development in Arabidopsis thaliana. Plant J 50:751–766CrossRefPubMedGoogle Scholar
  16. 16.
    Cardoza V, Stewart CN (2003) Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants. Plant Cell Rep 21:599–604PubMedGoogle Scholar
  17. 17.
    Sessions A, Weigel D, Yanofsky MF (1999) The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J 20:259–263CrossRefPubMedGoogle Scholar
  18. 18.
    Hadfield KA, Bennett AB (1998) Polygalacturonases: many genes in search of a function. Plant Physiol 117:337–343CrossRefPubMedGoogle Scholar
  19. 19.
    Torki M, Mandaron P, Thomas F, Quigley F, Mache R, Falconet D (1999) Differential expression of a polygalacturonase gene family in Arabidopsis thaliana. Mol Gen Genet 261:948–952CrossRefPubMedGoogle Scholar
  20. 20.
    Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucl Acids Res 30:325–327CrossRefPubMedGoogle Scholar
  21. 21.
    Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database. Nucl Acids Res 27:297–300CrossRefPubMedGoogle Scholar
  22. 22.
    Bate N, Twell D (1998) Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements. Plant Mol Biol 37:859–869CrossRefPubMedGoogle Scholar
  23. 23.
    Rogers H, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale D, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585CrossRefPubMedGoogle Scholar
  24. 24.
    Meshi T, Taoka KI, Iwabuchi M (2000) Regulation of histone gene expression during the cell cycle. Plant Mol Biol 43:643–657CrossRefPubMedGoogle Scholar
  25. 25.
    Van Der Straeten D, Rodrigues-Pousada RA, Goodman HM, Van Montagu M (1991) Plant enolase: gene structure, expression, and evolution. Plant Cell 3:719–735CrossRefPubMedGoogle Scholar
  26. 26.
    Gubler F, Jacobsen JV (1992) Gibberellin-responsive elements in the promoter of a barley high-pl a-amylase gene. Plant Cell 4:1435–1441CrossRefPubMedGoogle Scholar
  27. 27.
    Mena M, Vicente-Carbajosa J, Schmidt RJ, Carbonero P (1998) An endosperm-specific DOF protein from barley, highly conserved in wheat, binds to and activates transcription from the prolamin-box of a native B-hordein promoter in barley endosperm. Plant J 16:53–62CrossRefPubMedGoogle Scholar
  28. 28.
    Lin X, Feng XH, Watson JC (1991) Differential accumulation of transcripts encoding protein kinase homologs in greening pea seedlings. Proc Natl Acad Sci USA 88:6951–6955CrossRefPubMedGoogle Scholar
  29. 29.
    Taylor JE, Webb STJ, Coupe SA, Tucker GA, Roberts JA (1993) Changes in polygalacturonase activity and solubility of polyuronides during ethylene-stimulated leaf abscission in Sambucus nigra. J Exp Bot 44:93–98CrossRefGoogle Scholar
  30. 30.
    Bonghi C, Rascio N, Ramina A, Casadoro G (1992) Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach. Plant Mol Biol 20:839–848CrossRefPubMedGoogle Scholar
  31. 31.
    Meakin PJ, Roberts JA (1991) Anatomical and biochemical changes associated with the induction of oilseed rape (Brassica napus) pod dehiscence by Dasineura brassicae (Winn.). Ann Bot 67:193–197Google Scholar
  32. 32.
    Pressey R (1991) Polygalacturonase in tree pollens. Phytochemistry 30:1753–1755CrossRefGoogle Scholar
  33. 33.
    Pressey R, Reger BJ (1989) Polygalacturonase in pollen from corn and other grasses. Plant Sci 59:57–62CrossRefGoogle Scholar
  34. 34.
    Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224CrossRefPubMedGoogle Scholar
  35. 35.
    Paul W, Hodge R, Smartt S, Draper J, Scott R (1992) The isolation and characterization of the tapetum-specific Arabidopsis thaliana A9 gene. Plant Mol Biol 19:611–622CrossRefPubMedGoogle Scholar
  36. 36.
    Yokoi S, Tsuchiya T, Toriyama K, Hinata K (1997) Tapetum specific expression of the Osg6B promoter-β-glucuronidase gene in transgenic rice. Plant Cell Rep 16:363–367Google Scholar
  37. 37.
    Roberts MR, Foster GD, Blundell RP, Robinson SW, Kumar A, Draper J, Scott R (1993) Gametophytic and sporophytic expression of an anther-specific Arabidopsis thaliana gene. Plant J 3:111–120CrossRefPubMedGoogle Scholar
  38. 38.
    Xu HL, Davies SP, Kwan BYH, Obrien AP, Singh M, Knox RB (1993) Haploid and diploid expression of a Brassica campestris anther-specific gene promoter in Arabidopsis and tobacco. Mol Gen Genet 239:58–65PubMedGoogle Scholar
  39. 39.
    Shiba H, Takayama S, Iwano M, Shimosato H, Funato M, Nakagawa T, Che FS, Suzuki Go, Watanabe M, Hinata K, Isogai A (2001) A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species. Plant Physiol 125:2095–2103CrossRefPubMedGoogle Scholar
  40. 40.
    Gόmez MD, Beltrάn JP, Cañas LA (2004) The pea END1 promoter drives anther-specific gene expression in different plant species. Planta 219:967–981CrossRefGoogle Scholar
  41. 41.
    Roque E, Gomez MD, Ellul P, Wallbraun M, Madueno F, Beltran JP, Canas LA (2007) The PsEND1 promoter: a novel tool to produce genetically engineered male-sterile plants by early anther ablation. Plant Cell Rep 26:313–325CrossRefPubMedGoogle Scholar
  42. 42.
    Zhang Q, Liu HZ, Cao JS (2008) Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. Chinensis. Mol Biol Rep 35:685–691CrossRefPubMedGoogle Scholar
  43. 43.
    Mariani C, De Beuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature 347:737–741CrossRefGoogle Scholar
  44. 44.
    Höfig KP, Moyle RL, Putterill J, Walter C (2003) Expression analysis of four Pinus radiata male cone promoters in the heterologous host Arabidopsis. Planta 217:858–867CrossRefPubMedGoogle Scholar
  45. 45.
    Hong HP, Gerster JL, Datla RSS, Albani D, Scoles G, Keller W, Robert LS (1997) The promoter of a Brassica napus polygalacturonase gene directs pollen expression of β-glucuronidase in transgenic Brassica plants. Plant Cell Rep 16:373–378Google Scholar
  46. 46.
    Tebbutt SJ, Rogers JH, Lonsdale DM (1994) Characterization of a tobacco gene encoding a pollen-specific polygalacturonase. Plant Mol Biol 25:283–297CrossRefPubMedGoogle Scholar
  47. 47.
    Allen RL, Lonsdale DM (1993) Molecular characterization of one of the maize polygalacturonase gene family members which are expressed during late pollen development. Plant J 3:261–271CrossRefPubMedGoogle Scholar
  48. 48.
    González-Carranza ZH, Whitelaw CA, Swarup R, Roberts JA (2002) Temporal and spatial expression of a polygalacturonase during leaf and flower abscission in oilseed rape and Arabidopsis. Plant Physiol 128:534–543CrossRefPubMedGoogle Scholar
  49. 49.
    Ogawa M, Kay P, Wilson S, Swain SM (2009) ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 are polygalacturonases required for cell separation during reproductive development in Arabidopsis. Plant Cell 21:216–233CrossRefPubMedGoogle Scholar
  50. 50.
    Medford JI, Elmer JS, Klee HJ (1991) Molecular cloning and characterization of genes expressed in shoot apical meristems. Plant Cell 3:359–370CrossRefPubMedGoogle Scholar
  51. 51.
    De Silva J, Jarman CD, Arrowsmith DA, Stronach MS, Chengappa S, Sidebottom C, Reid JSG (1993) Molecular characterisation of a xyloglucan-specific endo-(1 → 4)-β-Dglucanase (xyloglucan endo-transglycosylase) from nasturtium seeds. Plant J. 3:701–711CrossRefPubMedGoogle Scholar
  52. 52.
    Nishitani K (1997) The role of endoxyloglucan transferase in organization of plant cell walls. Int Rev Cytol 173:157–206CrossRefPubMedGoogle Scholar
  53. 53.
    Nishitani K (1998) Construction and restructuring of cellulosexyloglucan framework in the apoplast as mediated by the xyloglucan-related protein family: A hypothetical scheme. J Plant Res 111:159–166CrossRefGoogle Scholar
  54. 54.
    Campbell P, Braam J (1999) Xyloglucan endotransglycosylases: diversity of genes, enzymes and potential wall-modifying functions. Trends Plant Sci 4:361–366CrossRefPubMedGoogle Scholar
  55. 55.
    Sasaki K, Hiraga S, Ito H, Seo S, Matsui H, Ohashi Y (2002) A wound-inducible tobacco peroxidase gene expresses preferentially in the vascular system. Plant Cell Physiol 43(1):108–117CrossRefPubMedGoogle Scholar
  56. 56.
    Sugimoto K, Takeda S, Hirochika H (2003) Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein AGP1 to the AG-motif (AGATCCAA) of the wound-inducible Myb gene NtMyb2. Plant J 36:550–564CrossRefPubMedGoogle Scholar
  57. 57.
    Kawaoka A, Kawamoto T, Sekine M, Yoshida K, Takano M, Sinmyo A (1994) A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene. Plant J 6:87–97CrossRefPubMedGoogle Scholar
  58. 58.
    Delessert C, Wilson IW, Van Der Straeten D, Dennis ES, Dolferus R (2004) Spatial and temporal analysis of the local response to wounding in Arabidopsis leaves. Plant Mol Biol 55:165–181CrossRefPubMedGoogle Scholar
  59. 59.
    Suzuki K, Suzuki N, Ohme-Takagi M, Shinshi H (1998) Immediate early induction of mRNAs for ethylene-responsive transcription factors in tobacco leaf strips after cutting. Plant J 15:657–665CrossRefGoogle Scholar
  60. 60.
    Nishiuchi T, Hamada T, Kodama H, Iba K (1997) Wounding changes the spatial expression pattern of the Arabidopsis plastid omega-3 fatty acid desaturase gene (FAD7) throughdifferent signal transduction pathways. Plant Cell 9:1701–1712CrossRefPubMedGoogle Scholar
  61. 61.
    Rushton PJ, Reinstadler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14:749–762CrossRefPubMedGoogle Scholar
  62. 62.
    Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206CrossRefPubMedGoogle Scholar
  63. 63.
    Takeda S, Sugimoto K, Otsuki H, Hirochika H (1999) A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors. Plant J 18:383–393CrossRefPubMedGoogle Scholar
  64. 64.
    Anthony LS, Gregg AH (2005) Systemic signaling in the wound response. Curr Opin Plant Biol 8:369–377CrossRefGoogle Scholar
  65. 65.
    Sasaki K, Hiroyuki Ito, Mitsuhara I, Hiraga S, Seo S, Matsui H, Ohashi Y (2006) A novel wound-responsive cis-element, VWRE, of the vascular system-specific expression of a tobacco peroxidase gene, tpoxN1. Plant Mol Biol 62:753–768CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Lili Wan
    • 1
  • Xiuyun Xia
    • 1
  • Dengfeng Hong
    • 1
  • Guangsheng Yang
    • 1
  1. 1.National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina

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