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Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

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Abstract

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-β-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

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References

  1. Altschuler M, Mascarenhas JP: Heat shock proteins and effects of heat shock in plants. Plant Mol Biol 1: 103–115 (1982).

    Google Scholar 

  2. Altschuler M, Mascarenhas JP: Transcription and translation of heat shock proteins in seedlings and developing seeds of soybean exposed to a gradual temperature increase. Plant Mol Biol 5: 291–297 (1985).

    Google Scholar 

  3. Barnett T, Altschuler M, McDaniel CN, Mascarenhas JP: Heat shock induced proteins in plant cells. Devel Genet 1: 331–340 (1980).

    Google Scholar 

  4. Baszczynski CL, Fallis L: Isolation and nucleotide sequence of a genomic clone encoding a new Brassica napus napin gene. Plant Mol Biol 14: 633–635 (1990).

    Google Scholar 

  5. Blumenthal CS, Batey IL, Bekes F, Wrigley CW, Barlow EWR: Gliadin genes contain heat-shock elements: possible relevance to heat-induced changes in grain quality. J Cereal Sci 11: 185–187 (1990).

    Google Scholar 

  6. Blumenthal CS, Barlow EW, Wrigley CW: Growth environment and wheat quality: the effect of heat stress on dough properties and gluten proteins. J Cereal Sci 18: 3–21 (1993).

    Google Scholar 

  7. Chrispeels MJ, Greenwood JS: Heat stress enhances phytohemagglutinin synthesis but inhibits its transport out of the endoplasmic reticulum. Plant Physiol 83: 778–784 (1987).

    Google Scholar 

  8. Chuong PV, Beversdorf WD: High frequency embryogenesis through isolated microspore culture in Brassica napus L. and B. carinata Braun. Plant Sci 39: 219–226 (1985).

    Google Scholar 

  9. Chuong PV, DesLauriers C, Kott LS, Beversdorf WD: Effects of donor genotype and bud sampling on microspore culture of Brassica napus. Can J Bot 66: 1653–1657 (1988).

    Google Scholar 

  10. Cooper P, Ho T-HD: Heat shock proteins in maize. Plant Physiol 71: 215–222 (1983).

    Google Scholar 

  11. Crouch ML: Non-zygotic embryos of Brassica napus L. contain embryo specific-storage proteins. Planta 156: 520–524 (1982).

    Google Scholar 

  12. Crouch ML, Tenbarge KM, Simon AE, Ferl R: cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor polypeptide. J Mol Appl Genet 2: 273–283 (1983).

    Google Scholar 

  13. DeLisle AJ, Crouch ML: Seed storage protein transcription and mRNA levels in Brassica napus during development and in response to exogenous abscisic acid. Plant Physiol 91: 617–623 (1989).

    Google Scholar 

  14. Dure L, Crouch M, Harada J, Ho T-HD, Mundy J, Quatrano R, Thomas T, Sung ZR: Common amino acid sequence domains among the LEA proteins of higher plants. Plant Mol Biol 12: 475–486 (1989).

    Google Scholar 

  15. Ericson ML, Rödin J, Lenman M, Glimelius K, Josefsson L-G, Rask L: Structure of the rapeseed 1.7S storage protein, napin, and its precursor. J Biol Chem 261: 14576–14581 (1986).

    Google Scholar 

  16. Ericson ML, Murén E, Gustavsson HO, Josefsson L-G, Rask L: Analysis of the promoter region of napin genes from Brassica napus demonstrates binding of nuclear proteins in vitro to a conserved sequence motif. Eur J Biochem 197: 741–746 (1991).

    Google Scholar 

  17. Fan Z, Armstrong KC, Keller WA: Development of microspores in vivo and in vitro in Brassica napus L. Protoplasma 147: 191–199 (1988).

    Google Scholar 

  18. Fernandez DE, Turner FR, Crouch ML: In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos. Development 111: 299–313 (1991).

    Google Scholar 

  19. Garrido D, Eller N, Heberle-Bors E, Vincente O: De novo transcription of specific mRNAs during the induction of tobacco pollen embryogenesis. Sex Plant Reprod 6: 40–45 (1993).

    Google Scholar 

  20. Gottlob-McHugh SG, Johnson DA: Detection of a subfamily of genes within the soybean nodulin-A multigene family. Can J Bot 69: 2663–2669 (1991).

    Google Scholar 

  21. Guerche P, Tire C, Grossi de Sa F, De Clerq A, Van Montagu M: Differential expression of the Arabidopsis 2S albumin genes and the effect of increasing gene family size. Plant Cell 2: 469–478 (1990).

    Google Scholar 

  22. Hause B, Hause G, Pechan P, Van Lammeren AAM: Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L. Cell Biol Int 17: 153–168 (1993).

    Google Scholar 

  23. Jefferson RA: Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).

    Google Scholar 

  24. Jefferson RA, Kavanagh TA, Bevan MW: GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 (1987).

    Google Scholar 

  25. Josefsson L-G, Lenman M, Ericson ML, Rask L: Structure of a gene encoding the 1.7S storage protein, napin, from Brassica napus. J Biol Chem 262: 12196–12201 (1987).

    Google Scholar 

  26. Keller WA, Fan Z, Pechan P, Long N, Grainger J: An efficient method for culture of isolated microspores of Brassica napus. In: Proceedings of the 7th International Rapeseed Congress (Poznan, Poland), vol 1, pp. 152–157 (1987).

  27. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K, Higashi K, Satoh S, Kamada H, Harada H: Isolation and characterization of a cDNA that encodes ECP31, an embryogenic-cell protein from carrot. Plant Mol Biol 19: 239–249 (1992).

    Google Scholar 

  28. Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K, Kamada H, Harada H. cDNA cloning of ECP40, an embryogenic-cell protein in carrot, and its expression during somatic and zygotic embryogenesis. Plant Mol Biol 21: 1053–1068 (1993).

    Google Scholar 

  29. Kosugi S, Ohashi Y, Nakajima K, Arai Y: An improved assay for β-glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous β-glucuronidase activity. Plant Sci 70: 133–140 (1990).

    Google Scholar 

  30. Kott LS, Polsoni L, Beversdorf WD: Cytological aspects of isolated microspore culture of Brassica napus. Can J Bot 66: 1658–1664 (1988).

    Google Scholar 

  31. Lehrach HD, Diamond D, Wozney JM, Boedtker H: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16: 4743–4751 (1977).

    Google Scholar 

  32. Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A laboratory Manual. Cold Spring Harbour Laboratory, Cold Spring Harbour, NY (1982).

    Google Scholar 

  33. Moloney MM, Walker JM, Sharma KK: High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8: 238–242 (1989).

    Google Scholar 

  34. Ouellet T, Rutledge RG, Miki B: Members of the acetohydroxyacid synthase multigene family of Brassica napus have divergent patterns of expression. Plant J 2: 321–330 (1992).

    Google Scholar 

  35. Pechan PM, Keller WA: Identification of potentially embryogenic microspores in Brassica napus. Physiol Plant 74: 377–384 (1988).

    Google Scholar 

  36. Pechan PM, Keller WA: Induction of microspore embryogenesis in Brassica napus L. by gamma irradiation and ethanol stress. In Vitro 25: 1073–1074 (1989).

    Google Scholar 

  37. Pechan PM, Bartels D, Brown DCW, Schell J: Messenger RNA and protein changes associated with induction of Brassica microspore embryogenesis. Planta 184: 161–165 (1991).

    Google Scholar 

  38. Raghavan V: Embryogenesis in Angiosperms: A Developmental and Experimental Study. Cambridge University Press, New York (1986).

    Google Scholar 

  39. Reynolds TL, Kitto SL: Identification of embryoid-abundant genes that are temporally expressed during pollen embryogenesis in wheat anther cultures. Plant Physiol 100: 1744–1750 (1992).

    Google Scholar 

  40. Scofield SR, Crouch ML: Nucleotide sequence of a member of the napin storage protein family from Brassica napus. J Biol Chem 262: 12202–12208 (1987).

    Google Scholar 

  41. Simon AE, Tenbarge KM, Scofield SR, Finkelstein RR, Crouch ML: Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum. Plant Mol Biol 5: 191–201 (1985).

    Google Scholar 

  42. Telmer CA, Simmonds DH, Newcomb W: Determination of developmental stage to obtain high frequencies of embryogenic microspores in Brassica napus. Physiol Plant 84: 417–424 (1992).

    Google Scholar 

  43. Telmer CA, Newcomb W, Simmonds D: Microspore development in Brassica napus and the effect of high temperature on division symmetry in vivo and in vitro. Protoplasma 172: 154–165 (1993).

    Google Scholar 

  44. Thomas PS: Hybridization of denatured RNA transferred or dotted onto nitrocellulose paper. Meth Enzymol 100: 255–267 (1983).

    Google Scholar 

  45. Vierling E: The roles of heat shock proteins in plants. Annu Rev Plant Physiol Mol Biol 42: 579–620 (1991).

    Google Scholar 

  46. Wiberg E, Råhlen L, Hellman M, Tillberg E, Glimelius K, Stymne S: The microspore-derived embryo of Brassica napus L. as a tool for studying embryo-specific lipid biogenesis and regulation of oil quality. Theor Appl Genet 82: 515–520 (1991).

    Google Scholar 

  47. Zaki MAM, Dickinson HG: Structural changes during the first divisions of embryos resulting from anther and free microspore culture in Brassica napus. Protoplasma 156: 149–162 (1990).

    Google Scholar 

  48. Zaki MAM, Dickinson HG: Microspore-derived embryos in Brassica: the significance of division symmetry in pollen mitosis I to embryogenic development. Sex Plant Reprod 4: 48–55 (1991).

    Google Scholar 

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Boutilier, K.A., Ginés, MJ., DeMoor, J.M. et al. Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis. Plant Mol Biol 26, 1711–1723 (1994). https://doi.org/10.1007/BF00019486

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  • DOI: https://doi.org/10.1007/BF00019486

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