Plant Molecular Biology

, Volume 57, Issue 6, pp 835–853 | Cite as

The Oryza sativa no pollen (Osnop) gene plays a role in male gametophyte development and most likely encodes a C2-GRAM domain-containing protein

  • Shu Ye Jiang
  • Minnie Cai
  • Srinivasan Ramachandran


Phenotype screens of Ds insertional lines identified a male sterile Orysa sativa no pollen (Osnop) mutant with a pollen-less phenotype at the flowering stage. The mutant phenotype showed linkage to Ds insertion into Osnop gene region. This mutant contained a deletion of 65 kb chromosomal region at the site of Ds insertion containing 14 predicted genes. Out of these deleted genes, Delegen 5–7, 9–10 were redundant, as two or three copies were present with 100% homology in other regions of rice genome. RT-PCR analysis showed that Delegen 5–7 were expressed not only in wild type plants but also in the mutant plants. In addition to this, Delegen 8–10 transcripts could not be detected under normal growth conditions, and Delegen 12 was expressed only in roots, thus deletion of these genes may not affect the pollen development. Our data and analysis also ruled out the possibility of delegen 1–4, 11, and 13 as candidates contributing to the pollen-less phenotype. Further investigation showed that the delegen 14 was expressed only in late stage of pollen development with the highest expression at the stage of pollen release and germination by RT-PCR, Northern blotting, in situ hybridization, and promoter-GUS transgenic plants. Thus, the delegen 14 gene is the best candidate for Osnop, corresponding to the pollen-less phenotype in the mutant. Our data suggest that delegen 14 may play an important role during late stage of pollen development and its germination. Since the delegen 14 gene has both C2 and GRAM domains, it can be assumed that this gene cross-links both calcium and phosphoinositide signaling pathways. This is the first report to suggest possible functions for this gene in plant development.


C2-GRAM domain deletion mutant Ds gametophyte development Oryza sativa 



flanking sequence tag


glucosyltransferases, Rab-like GTPase activators and myotubularin




Oryza sativa no pollen


rapid amplification of cDNA ends


reverse transcription PCR


scanning electron microscopy


thermal asymmetric interlaced-PCR


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Albani, D., Robert, L.S, Donaldson, P.A, Altosaar, I., Arnison, P.G., Fabijanski, S.F. 1990Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore developmentPlant Mol. Biol.15605622CrossRefPubMedGoogle Scholar
  2. Ariizumi, T., Hatakeyama, K., Hinata, K., Inatsugi, R., Nishida, I., Sata, S., Kato, T., Tabata, S., Toriyama, K. 2004Disruption of the novel plant protein NEF1 affects lipid accumulation in the plastids of the tapetum and exine formation of pollen., resulting in male sterility in Arabidopsis thalianaPlant J.39170181CrossRefGoogle Scholar
  3. Bahrami, A.R., Gray, J.E. 1998Expression of a proteasome alpha-type subunit gene during tobacco development and senescencePlant Mol. Biol.39325333CrossRefGoogle Scholar
  4. Bate, N., Twell, D. 1998Functional architecture of a late pollen promoter, pollen-specific transcription is developmentally regulated by multiple stage-specific and codependent activator elementsPlant Mol. Biol.37859869CrossRefPubMedGoogle Scholar
  5. Becker, J., Boavida, L.C., Carneiro, J., Haury, M., Feijó, J.A. 2003Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptomePlant Physiol.133713725CrossRefPubMedGoogle Scholar
  6. Blanc, G., Wolfe, K.H. 2004Functional divergence of duplicated genes formed by polyploidy during Arabidopsis evolutionPlant Cell1616791691CrossRefPubMedGoogle Scholar
  7. Bucciaglia, P.A., Smith, A.G. 1994Cloning and characterization of Tag 1., a tobacco anther beta-1, 3-glucanase expressed during tetrad dissolutionPlant Mol. Biol.24903914CrossRefPubMedGoogle Scholar
  8. Bucciaglia, P.A., Zimmermann, E., Smith, A.G. 2003Functional analysis of a β-1., 3-glucanase gene Tag 1. with anther-specific RNA and protein accumulation using antisense RNA inhibitionJ. Plant Physiol.16013671373PubMedGoogle Scholar
  9. Clark, J.D., Lin, L.L., Kriz, R.W., Ramesha, C.S., Sultzman, L.A., Lin, AY., Milona, N., Knopf, J.L. 1991A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAPCell6510431051PubMedGoogle Scholar
  10. Custers, J.B.M., Oldenhof, M.T., Schrauwen, J.A.M., Cordewener, J.H.G., Wullems, G.J., Campagne, M.M.L. 1997Analysis of microspore-specific promoters in transgenic tobaccoPlant Mol. Biol.35689699PubMedGoogle Scholar
  11. Dellaporta, S.L., Wood, J., Hicks, J.B. 1983A plant DNA minipreparation, version 2Plant Mol. Biol. Rep.11922Google Scholar
  12. Ding, Z.J., Wu, X.H., Wang, T 2002The rice tapetum-specific gene RA39 encodes a type I ribosome-inactivating proteinSex Plant Reprod.15205212Google Scholar
  13. Doerks, T., Strauss, M., Brendel, M., Bork, P. 2000GRAM., a novel domain in glucosyltransferases., myotubularins and other putative membrane-associated proteinsTrends Biochem. Sci.25483485PubMedGoogle Scholar
  14. Filichkin, S.A., Leonard, J.M., Monteros, A., Liu, P.P., Nonogaki, H. 2004A novel endo-β-mannanase gene in tomato LeMAN5 is associated with anther and pollen developmentPlant Physiol.13410801087PubMedGoogle Scholar
  15. Franklin-Tong, V.E., Drobak, B.K., Allan, A.C., Watkins, P., Trewavas, A.J. 1996Growth of pollen tubes of Papaver rhoeas is regulated by a slow-moving calcium wave propagated by inositol 1.,4.,5-trisphosphatePlant Cell813051321PubMedGoogle Scholar
  16. Frohman, M.A., Dush, M.K., Marlin, G.R. 1988Rapid amplification of full-length cDNAs from rare transcripts, amplification using a single-specific oligonucleotide primersProc. Natl. Acad. Sci. USA8589989002PubMedGoogle Scholar
  17. Goff, S.A., Ricke, D., Lan, T.H., Presting, G., Wang, R., Dunn, M., Glazebrook, J., Sessions, A., Oeller, P., Varma, H., Hadley, D., Hutchison, D., Martin, C., Katagiri, F., Lange, B.M., Moughamer, T., Xia, Y., Budworth, P., Zhong, J., Miguel, T., Paszkowski, U., Zhang, S., Colbert, M., Sun, W., Chen, L., Cooper, B., Park, S., Wood, T.C., Mao, L., Quail, P., Wing, R., Dean, R., Yu, Y., Zharkikh, A., Shen, R., Sahasrabudhe, S., Thomas, A., Cannings, R., Gutin, A., Pruss, D., Reid, J., Tavtigian, S., Mitchell, J., Eldredge, G., Scholl, T., Miller, R.M., Bhatnagar, S., Adey, N., Rubano, T., Tusneem, N., Robinson, R., Feldhaus, J., Macalma, T., Oliphant, A., Briggs, S. 2002A Draft sequence of the rice genome Oryza sativa L. ssp.japonicaScience29692100CrossRefPubMedGoogle Scholar
  18. Goldberg, R.B., Beals, T.P., Sanders, P.M. 1993Anther development, basic principles and practical applicationsPlant Cell512171229Google Scholar
  19. Guerineau, F., Sorensen, A.M., Fenby, N., Scott, R.J. 2003Temperature sensitive diphtheria toxin confers conditional male-sterility in Arabidopsis thalianaPlant Biotech. J.13342Google Scholar
  20. Hanson, D.D., Hamilton, D.A., Travis, J.L., Bashe, D.M., Mascarenhas, J.P. 1989Characterization of a pollen-specific cDNA clone from Zea mays and its expressionPlant Cell1173179PubMedGoogle Scholar
  21. Hihara, Y., Hara, C., Uchimiya, H. 1996Isolation and characterization of two cDNA clones for mRNAs that are abundantly expressed in immature anthers of rice Oryza sativa LPlant Mol. Biol.3011811193PubMedGoogle Scholar
  22. Hirochika, H. 2001Contribution of the Tos17 retrotransposon to rice functional genomicsCurr. Opin. Plant Biol.4118122PubMedGoogle Scholar
  23. Hirochika, H., Sugimoto, K., Otsuki, Y., Tsugawa, H., Kanda, M. 1996Retrotransposons of rice involved in mutations induced by tissue cultureProc. Natl. Acad. Sci. USA9377837788PubMedGoogle Scholar
  24. Honys, D., Twell, D. 2003Comparative analysis of the Arabidopsis pollen transcriptomePlant Physiol.132640652PubMedGoogle Scholar
  25. Ichihara, K., Murota, N., Fujii, S. 1990Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds, a possible mechanism of feedforward control of triacylglycerol synthesis by fatty acidsBiocheim. Biophys. Acta1043227234Google Scholar
  26. Jefferson, R.A. 1987Assaying chimeric genes in Plants, The GUS gene fusion systemPlant Mol. Biol. Report.5387405Google Scholar
  27. Jeon, J.S., Chung, Y.Y., Lee, S., Yi, G.H., Oh, B.G., An, G. 1999Isolation and characterization of an anther-specific gene., RA8., from rice Oryza sativa LPlant Mol. Biol.393544PubMedGoogle Scholar
  28. Jouannic, N., Lepetit, M., Vergnolle, C., Cantrel, C., Gardies, A.M., Kader, J.C., Arondel, V. 1998Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeastEur. J. Biochem.258402410PubMedGoogle Scholar
  29. Kamalay, J.C., Goldberg, R.B. 1980Regulation of structural gene expression in tobaccoCell19934946Google Scholar
  30. Kaya, H., Sato, S., Tabata, S., Kobayashi, Y., Iwabuchi, M., Araki, T. 2000Hosoba toge toge., a syndrome caused by a large chromosomal deletion associated with T-DNA insertion in ArabidopsisPlant Cell Physiol.4110551066PubMedGoogle Scholar
  31. Kim, K.C., Hur, Y., Maeng, J. 2003Isolation of a gene., PnFL-1., expressed in Pharbitis cotyledons during floral inductionMol. Cells165459PubMedGoogle Scholar
  32. Kocsis, M.G., Weselake, R.J. 1996Phosphatidate phosphatases of mammals., yeast., and higher plantsLipids31785802PubMedGoogle Scholar
  33. Kolesnik, T., Szeverenyi, I., Bachmann, D., Kumar, C.S., Jiang, S., Ramamoorthy, R., Cai, M., Ma, Z.G., Sundaresan, V., Ramachandran, S. 2004Establishing an efficient Ac/Ds tagging system in rice, large-scale analysis of Ds flanking sequencesPlant J.37301314PubMedGoogle Scholar
  34. Lalanne, E., Honys, D., Johnson, A., Borner, G.H.H., Lilley, K.S., Dupree, P., Grossniklaus, U., Twell, D. 2004SETH1 and SETH2, two components of the glycosylphosphatidylinositol anchor biosynthetic pathway, are required for pollen germination and tube growth in arabidopsisPlant Cell16229240PubMedGoogle Scholar
  35. Liu, Y.G., Whittier, R.F. 1995Thermal asymmetric interlaced PCR, automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walkingGenomics25674681PubMedGoogle Scholar
  36. Lorrain, S., Lin, B., Auriac, M.C., Kroj, T., Saindrenan, P., Nicole, M., Balague, C., Robya, D. 2004VASCULAR ASSOCIATED DEATH1, a novel GRAM domain-containing protein, is a regulator of cell death and defense responses in vascular tissuesPlant Cell1622172232PubMedGoogle Scholar
  37. Ludwig, S.R., Oppenheimer, D.G., Silflow, C.D., Snudstad, D.P. 1998The α1-tubulin gene in Arabidopsis thaliana, primary structure and preferential expression in flowersPlant Mol. Biol.10311321Google Scholar
  38. Lynch, M., Conery, J.S. 2000The evolutionary fate and␣consequences of duplicate genesScience29011511155CrossRefPubMedGoogle Scholar
  39. Marcel, G.C., Matos, A., darcy-Lameta, A., Kader, J.C., Zuily-Fodil, Y., Pham-Thi, A. 2000Two novel plant cDNAs homologous to animal type-2 phosphatidate phosphatase are expressed in cowpea leaves and are differently regulated by water deficitsBiochem. Soc. Trans.28915917PubMedGoogle Scholar
  40. Mariani, C., Beuckeleer, M.D., Truettner, J., Leemans, J., Goldberg, R.B. 1990Induction of male sterility in plants by a chimaeric ribonuclease geneNature347737741Google Scholar
  41. McCormick, S. 2004Control of male gametophyte developmentPlant Cell16S142S153CrossRefPubMedGoogle Scholar
  42. Mogami, N., Shiota, H., Tanaka, I. 2002LP28., a lily pollen-specific LEA-like protein., is located in the callosic cell wall during male gametogenesisSex. Plant Reprod.155763Google Scholar
  43. Nacry, P., Camilleri, C., Courtial, B., Caboche, M., Bouchez, D. 1998Major chromosomal rearrangements induced by T-DNA transformation in ArabidopsisGenetics149641650PubMedGoogle Scholar
  44. Nonomura, K.I., Miyoshi, K., Eiguchi, M., Suzuki, T., Miyao, A., Hirochika, H., Kurata, N. 2003The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in ricePlant Cell1517281739PubMedGoogle Scholar
  45. Nonomura, K.I., Nakano, M., Fukuda, T., Eiguchi, M., Miyao, A., Hirochika, H., Kurata, N. 2004aThe novel gene HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 of rice encodes a putative coiled-coil protein required for homologous chromosome pairing in meiosisPlant Cell1610081020Google Scholar
  46. Nonomura, K.I., Nakano, M., Murata, K., Miyoshi, K., Eiguchi, M., Miyao, A., Hirochika, H., Kurata, N. 2004bAn insertional mutation in the rice PAIR2 gene., the ortholog of Arabidopsis ASY1., results in a defect in homologous chromosome pairing during meiosisMol. Genet. Genomics271121129Google Scholar
  47. Oh, S.A., Park, S.K., Jang, I., Howden, R., Moore, J.M., Grossniklaus, U., Twell, D. 2003Halfman., an Arabidopsis male gametophytic mutant associated with a 150 kb chromosomal deletion adjacent to an introduced Ds transposable elementSex. Plant Reprod.1699102Google Scholar
  48. Oku, M., Warnecke, D., Noda, T., Muller, F., Heinz, E., Mukaiyama, H., Kato, N., Sakai, Y. 2003Perxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domainEMBO J.2232313241PubMedGoogle Scholar
  49. Oldenhof, M.T., Groot, P.F.M., Visser, J.H., Schrauwen, J.A.M., Wullems, G.J. 1996Isolation and characterization of a microspore-specific gene from tobaccoPlant Mol. Biol.31213225PubMedGoogle Scholar
  50. Page, D.R., Grossniklaus, U. 2002The art and design of genetic screens, Arabidopsis thalianaNat. Rev. Genet.3124136PubMedGoogle Scholar
  51. Page, D.R., Kohler, C., da Costa-Nunes, J.A., Baroux, C., Moore, J.M., Grossniklaus, U. 2004Intrachromosomal excision of a hybrid Ds element induces large genomic deletions in ArabidopsisProc. Natl. Acad. Sci. USA10129692974PubMedGoogle Scholar
  52. Peters, J.M. 1994Proteasomes, protein degradation machines of the cellTrends Biochem. Sci.19377382PubMedGoogle Scholar
  53. Ponting, C.P., Parker, P.J. 1996Extending the C2 domain family, C2s in PKCs delta., epsilon., eta., theta., phospholipases., GAPs., and perforinProtein Sci.5162166PubMedGoogle Scholar
  54. Ramachandran, S., Sundaresan, V. 2001Transposons as tools for functional genomicsPlant Physiol. Biochem.39243252Google Scholar
  55. Sambrook, J., Fritsch, E.F., Maniayis, T. 1989Molecular cloning, A laboratory manual2nd edCold Spring Harbor Laboratory PressCold Spring Harbor., NYGoogle Scholar
  56. Sanders, P.M., Bui, A.Q., Weterings, K., McIntire, K.N., Hsu, Y.C., Lee, P.Y., Truong, M.T., Beals, T.P., Goldberg, R.B. 1999Anther developmental defects in Arabidopsis thaliana male-sterile mutantsSex. Plant Reprod.11297322Google Scholar
  57. Schneidereit, A., Scholz-Starke, J., Buttner, M. 2003Functional characterization and expression analyses of the glucose-specific AtSTP9 monosaccharide transporter in pollen of ArabidopsisPlant Physiol.133182190PubMedGoogle Scholar
  58. Scott, R.J., Spielman, M., Dickinsonb, H.G 2004Stamen structure and functionPlant Cell16S46S60Google Scholar
  59. Shen, J.B., Hsu, F.C. 1992Brassica anther-specific genes, characterization and in situ localization of expressionMol. Gen. Genet.234379389PubMedGoogle Scholar
  60. Speranza, A., Scossianti, V., Crinelli, R., Calzoni, G.L., Magnani, M. 2001Inhibition of proteasome activity strongly affects kiwifruit pollen germination. Involvement of the ubiquitin/ proteasome pathway as a major regulatorPlant Physiol.12611501161PubMedGoogle Scholar
  61. Suen, D.F., Wu, S.S.H., Chang, H.C., Dhugga, K.S., Huang, A.H.C. 2003Cell wall reactive proteins in the coat and wall of maize pollenJ. Biol. Chem.2784367243681PubMedGoogle Scholar
  62. Tax, F.E., Vernon, D.M. 2001T-DNA-associated duplication/translocations in Arabidopsis. implications for mutant analysis and functional genomicsPlant Physiol.12615271538PubMedGoogle Scholar
  63. Thomas, J.H. 1993Thinking about genetic redundancyTrends Genet.9395399PubMedGoogle Scholar
  64. Tsuchiya, T., Toriyama, K., Ejiri, S., Hinata, K. 1994Molecular characterization of rice genes specifically expressed in the anther tapetumPlant Mol. Biol.2617371746PubMedGoogle Scholar
  65. Tsuchiya, T., Toriyama, K., Yoshikawa, M., Ejiri, S., Hinata, K. 1995Tapetum-specific expression of the gene for an endo-beta-1., 3-glucanase causes male sterility in transgenic tobaccoPlant Cell Physiol.36487494PubMedGoogle Scholar
  66. Tsujita, K., Itoh, T., Ijuin, T., Yamamoto, A., Shisheva, A., Laporte, J., Takenawa, T. 2004Myotubularin regulates the function of the late endosome through the GRAM domain-phosphatidylinositol 3., 5-bisphosphate interactionJ. Biol. Chem.2791381713824PubMedGoogle Scholar
  67. Twell, D., Wing, R., Yamaguchi, J., McCormick, S. 1989Isolation and expression of an anther-specific gene from tomatoMol. Gen. Genet.217240245PubMedGoogle Scholar
  68. Twell, D., Yamaguchi, J., McCormick, S. 1990Pollen-specific gene expression in transgenic plants, coordinate regulation of two different tomato gene promoters during microsporogenesisDevelopment109705713PubMedGoogle Scholar
  69. Ursin, V.M., Yamaguchi, J., McCormick, S. 1989Gametophytic and sporophytic expression of anther-specific genes in developing tomato anthersPlant Cell1727736PubMedGoogle Scholar
  70. Wang, S.H., Chen, F., Zhou, K.D. 2000In vitro pollen germination of riceActa Agro. Sinica26609612(in Chinese)Google Scholar
  71. Willing, R.P., Bashe, D., Mascarenhas, J.P. 1988An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea maysTheor. Appl. Genet.75751753Google Scholar
  72. Willing, R.P., Mascarenhas, J.P. 1984Analysis of the complexity and diversity of mRNAs from pollen and shoots of TradescantiaPlant Physiol.75865868Google Scholar
  73. Worrall, D., Hird, D.L., Hodge, R., Paul, W., Draper, J., Scott, R. 1992Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobaccoPlant Cell.4759771PubMedGoogle Scholar
  74. Xu, H., Theerakulpisut, P., Taylor, P.E., Knox, R.B., Singh, M.B., Bhalla, P.L. 1995Isolation of a gene preferentially expressed in mature anthers of rice Oryza sativa LProtoplasma187127131Google Scholar
  75. Yokoi, S., Tsuchiya, T., Toriyama, K., Hinata, K. 1997Tapetum-specific expression of the Osg6B promoter-β-glucuronidase gene in transgenic ricePlant Cell Rep.16363367Google Scholar
  76. Zhan, X.Y., Wu, H.M., Cheung, A.Y. 1996Nuclear male sterility induced by pollen-specific expression of a ribonucleaseSex. Plant Reprod.93543Google Scholar
  77. Zhu, Q.H., Ramm, K., Shivakkumar, R., Dennis, E.S., Upadhyaya, N.M. 2004The ANTHER INDEHISCENCE1 gene encoding a single MYB domain protein is involved in anther development in ricePlant Physiol.13515141525PubMedGoogle Scholar
  78. Zou,  1994Characterization of a rice pollen-specific gene and its expressionAm. J. Bot.81552561Google Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Shu Ye Jiang
    • 1
  • Minnie Cai
    • 1
  • Srinivasan Ramachandran
    • 1
  1. 1.Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research LinkNational University of SingaporeSingapore

Personalised recommendations