Advertisement

Plant Molecular Biology

, Volume 55, Issue 5, pp 715–726 | Cite as

Silencing of the pollen-specific gene NTP303 and its family members in tobacco affects in vivo pollen tube growth and results in male sterile plants

  • Peter de Groot
  • Koen Weterings
  • Mark de Been
  • Floyd Wittink
  • Raymond Hulzink
  • Jan Custers
  • Marinus van Herpen
  • George Wullems
Article

Abstract

In seed plants, successful fertilization requires correct regulation of pollen tube growth. At germination and during growth, the pollen tube interacts with tissues from the pistil while the pollen tube extends via tip growth. Despite the fact that much research has been devoted to the mechanisms regulating pollen tube growth, many aspects are currently unknown. Previously, we have isolated a pollen-specific gene from tobacco—NTP303—that probably functions during pollen tube growth. NTP303 is part of a family of five members. Its expression is regulated both at the transcriptional and at the translational level. While NTP303 transcripts accumulate to high levels between early bi-cellular and mature pollen stages, NTP303 protein is hardly detectable until germination and pollen tube growth. In order to elucidate the role and function of NTP303 in the pollen tube, we studied the effect of NTP303 gene silencing on pollen function. Therefore, we have transformed tobacco plants with NTP303 co-suppression and anti-sense gene constructs. In these plants, the kanamycin resistance trait—which was linked to the NTP303-silencing gene—was not transmitted through the male gametophyte. This indicated that lowering the transcript level of NTP303 and/or its family members interferes with pollen function. Because we could not find a readily distinguishable phenotype in pollen from the hemizygous anti-sense and co-suppression plants, we rescued the defective pollen to produce doubled haploid plants that were homozygous for the NTP303 anti-sense gene. We found that in pollen from these plants the transcript levels of all NTP303 family members were reduced. Although pollen and pollen tubes from these plants appeared completely normalin vitro, the pollen tubes showed slower growth rates in vivo and arrested in the style before they reached the ovary, so that fertilization failed. These data demonstrate that NTP303 and its family members are essential for normal pollen tube growth and indicate several possible functions.

Anti-sense expression Co-suppression Doubled haploid Pollen tube growth Tobacco 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Albani, D., Sardana, R., Robert, L.S., Altosaar, I., Arnison, P.G. and Fabijanski, S.F. 1992. A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen – molecular characterization and analysis of promoter activity in transgenic tobacco plants. Plant J. 2: 331–342.Google Scholar
  2. Alonso, J.M., Stepanova, A.N., Leisse, T.J., Kim, C.J., Chen, H.M., Shinn, P., Stevenson, D.K., Zimmerman, J., Barajas, P., Cheuk, R., Gadrinab, C., Heller, C., Jeske, A., Koesema, E., Meyers, C.C., Parker, H., Prednis, L., Ansari, Y., Choy, N., Deen, H., Geralt, M., Hazari, N., Hom, E., Karnes, M., Mulholland, C., Ndubaku, R., Schmidt, I., Guzman, P., Aguilar-Henonin, L., Schmid, M., Weigel, D., Carter, D.E., Marchand, T., Risseeuw, E., Brogden, D., Zeko, A., Crosby, W.L., Berry, C.C. and Ecker, J.R. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.Google Scholar
  3. Becker, J.D., Boavida, L.C., Carneiro, J., Haury, M. and Feijo, J.A. 2003. Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome. Plant Physiol. 133: 713–725.Google Scholar
  4. Bernatzky, R. and Tanksley, S.D. 1986. Genetics of actin related sequences in tomato. Theor. Appl. Genet. 72: 314–321.Google Scholar
  5. Bots, M., Feron, R., Uehlein, N., Weterings, K., Kaldenhoff, R. and Mariani, C. 2004. J. Exp. Bot., in press.Google Scholar
  6. Custers, J.B.M., Snepvangers, S., Jansen, H.J., Zhang, L. and van Lookeren Campagne, M.M. 1999. The 35S-CaMV promoter is silent during early embryogenesis but activated during non-embryogenic sporophytic development in microspore culture. Protoplasma 208: 257–264.Google Scholar
  7. Felsenstein, J. 1989. PHYLIP – Phylogeny Inference Package (Version 3.2). Cladistics 5: 164–166.Google Scholar
  8. Fidlerova, A., Smykal, P., Tupy, J. and Capkova, V. 2001. Glycoproteins 66 and 69 kDa of pollen tube wall: properties and distribution in angiosperms. J. Plant Physiol. 158: 1367–1374.Google Scholar
  9. Gupta, R., Ting, J.T.L., Sokolov, L.N., Johnson, S.A. and Luan, S. 2002. A tumor suppressor homolog, AtPTEN1, is essential for pollen development in Arabidopsis. Plant Cell 14: 2495–2507.Google Scholar
  10. Hepler, P.K., Vidali, L. and Cheung, A.Y. 2001. Polarized cell growth in higher plants. Ann Rev. Cell Dev. Biol. 17: 159–187.Google Scholar
  11. Holden, M.J., Marty, J.A. and Singh Cundy, A. 2003. Pollination-induced ethylene promotes the early phase of pollen tube growth in Petunia inflate. J. Plant Physiol. 160: 261–269.Google Scholar
  12. Honys, D. and Twell, D. 2003. Comparative analysis of the Arabidopsis pollen transcriptome. Plant Physiol. 132: 640–652.Google Scholar
  13. Honys, D., Combe, J.P., Twell, D. and Capkova, V. 2000. The translationally repressed pollen-specific NTP303 mRNA is stored in non-polysomal mRNPs during pollen maturation. Sex Plant Reprod. 13: 135–144.Google Scholar
  14. Horsch, R.B., Fry, J., Hoffman, N., Eichholtz, D., Rogers, S. and Fraley, R. 1985. A simple and general method for transferring genes into plants. Science 227: 1229–1231.Google Scholar
  15. Hulzink, R.J.M., de Groot, P.F.M., Croes, A.F., Quaedvlieg, W., Twell, D., Wullems, G.J. and van Herpen, M.M.A. 2002. The 5¢-untranslated region of the NTP303 gene strongly enhances translation during pollen tube growth, but not during pollen maturation. Plant Physiol. 129: 342–353.Google Scholar
  16. Kato, N. and Esaka, M. 1999. Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells. Physiol. Plant 105: 321–329.Google Scholar
  17. Kato, N. and Esaka, M. 2000. Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts. Planta 210: 1018–1022.Google Scholar
  18. Linskens, H.F. and Esser, K. 1957. U¨ ber eine spezifische Anfa¨ rbung der Pollenschla¨ uche im Griffel und die Zahl der Kallosepropfen nach Selbstung und Fremdung. Naturwissenschaften 44: 16–17.Google Scholar
  19. Lord, E.M. and Russell, S.D. 2002. The mechanisms of pollination and fertilization in plants. Ann Rev. Cell Dev. Biol. 18: 81–105.Google Scholar
  20. McCormick, S. 2004. Control of male gametophyte development. Plant Cell 16, S142–153.Google Scholar
  21. Moutinho, A., Camacho, L., Haley, A., Pais, M.S., Trewavas, A. and Malho´, R. 2001. Antisense perturbation of protein function in living pollen tubes. Sex Plant Reprod. 14: 101–104.Google Scholar
  22. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–479.Google Scholar
  23. Muschietti, J., Dircks, L., Vancanneyt, G. and McCormick, S. 1994. LAT52 protein is essential for tomato pollen development: pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization. Plant J. 6: 321–338.Google Scholar
  24. Read, S.M., Clarke, A.E. and Bacic, A. 1993. Stimulation of growth of cultured Nicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu-(II) salts. Protoplasma 177: 1–14.Google Scholar
  25. Russell, S.D. and Dumas, C. 1992. Sexual Reproduction in Flowering Plants. Academic Press, San Diego.Google Scholar
  26. Sanchez, A.M., Bosch, M., Bots, M., Nieuwland, J., Feron, R. and Mariani, C. 2004. Pistil factors controlling pollination. Plant Cell 16, S98–106.Google Scholar
  27. Schrauwen, J.A.M., de Groot, P.F.M., van Herpen, M.M.A., van der Lee, T., Reijnen, W.H., Weterings, K.A.P. and Wullems, G.J. 1990. Stage-related expression of mRNAs during pollen development in lily and tobacco. Planta 182: 298–304.Google Scholar
  28. Sedbrook, J.C., Carroll, K.L., Hung, K.F., Masson, P.H. and Somerville, C.R. 2002. The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth. Plant Cell 14: 1635–1648.Google Scholar
  29. Thompson, J.D., Higgins, D.G. and Gibson, T.J. 1994. Clustal-W-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucl. Acids Res. 22: 4673–4680.Google Scholar
  30. van Eldik, G.J., Vriezen, W.H., Wingens, M., Ruiter, R.K., van Herpen, M.M.A., Schrauwen, J.A.M. and Wullems, G.J. 1995. A pistil-specific gene of Solanum tuberosum is predominantly expressed in the stylar cortex. Sex Plant Reprod. 8: 173–179.Google Scholar
  31. Wang, M.B. and Waterhouse, P.M. 2002. Application of gene silencing in plants. Curr. Opin. Plant Biol. 5: 146–150.Google Scholar
  32. Weterings, K. and Russell, S.D. 2004. Experimental analysis of the fertilization process. Plant Cell 16, S107–118.Google Scholar
  33. Weterings, K., Reijnen, W., van Aarssen, R., Kortstee, A., Spijkers, J., Herpen, M., Schrauwen, J. and Wullems, G. 1992. Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination. Plant Mol. Biol. 18: 1101–1111.Google Scholar
  34. Weterings, K., Reijnen, W., Wijn, G., Heuvel, K., Appeldoorn, N., de Kort, G., van Herpen, M., Schrauwen, J. and Wullems, G. 1995. Molecular characterization of the pollenspecific genomic clone NTPg303 and in situ localization of expression. Sex Plant Reprod. 8: 11–17.Google Scholar
  35. Wittink, F.R.A., Knuiman, B., Derksen, J., Capkova, V., Twell, D., Schrauwen, J.A.M. and Wullems, G.J. 2000. The pollen-specific gene NTP303 encodes a 69-kDa glycoprotein associated with the vegetative membranes and the cell wall. Sex Plant Reprod. 12: 276–284.Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Peter de Groot
    • 1
  • Koen Weterings
    • 1
  • Mark de Been
    • 1
  • Floyd Wittink
    • 1
  • Raymond Hulzink
    • 1
  • Jan Custers
    • 2
  • Marinus van Herpen
    • 1
  • George Wullems
    • 1
  1. 1.Department of Experimental BotanyUniversity of NijmegenNetherlands
  2. 2.Plant Research International, TheNetherlands

Personalised recommendations