Plant Cell Reports

, Volume 27, Issue 11, pp 1741–1754 | Cite as

Efficient production of genetically engineered, male-sterile Arabidopsis thaliana using anther-specific promoters and genes derived from Brassica oleracea and B. rapa

  • Ken-ichi Konagaya
  • Sugihiro Ando
  • Shinichiro Kamachi
  • Mai Tsuda
  • Yutaka Tabei
Genetic Transformation and Hybridization


Prevention of transgene flow from genetically modified crops to food crops and wild relatives is of concern in agricultural biotechnology. We used genes derived from food crops to produce complete male sterility as a strategy for gene confinement as well as to reduce the food purity concerns of consumers. Anther-specific promoters (A3, A6, A9, MS2, and MS5) were isolated from Brassica oleracea and B. rapa and fused to the β-glucuronidase (GUS) reporter gene and candidate genes for male sterility, including the cysteine proteases BoCysP1 and BoCP3, and negative regulatory components of phytohormonal responses involved in male development. These constructs were then introduced into Arabidopsis thaliana. GUS analyses revealed that A3, A6, and A9 had tapetum-specific promoter activity from the anther meiocyte stage. Male sterility was confirmed in tested constructs with protease or gibberellin insensitive (gai) genes. In particular, constructs with BoCysP1 driven by the A3 or A9 promoter most efficiently produced plants with complete male sterility. The tapetum and middle layer cells of anthers expressing BoCysP1 were swollen and excessively vacuolated when observed in transverse section. This suggests that the ectopic expression of cysteine protease in the meiocyte stage may inhibit programmed cell death. The gai gene also induced male sterility, although at a low frequency. This is the first report to show that plant cysteine proteases and gai from food crops are available as a novel tool for the development of genetically engineered male-sterile plants.


Male sterility Tapetum Cysteine protease Gene flow Programmed cell death 



Cauliflower mosaic virus


Cytokinin oxidase/dehydrogenase




Genetically modified




Open reading frame


Polymerase chain reaction


Rapid amplification of cDNA ends


Programmed cell death


Thermal asymmetric interlaced PCR

Supplementary material

299_2008_598_MOESM1_ESM.bmp (27.8 mb)
Supplementary figure S1 (BMP 28476 kb)
299_2008_598_MOESM2_ESM.bmp (61.8 mb)
Supplementary figure S2 (BMP 63245 kb)


  1. Aarts MG, Dirkse WG, Stiekema WJ, Pereira A (1993) Transposon tagging of a male sterility gene in Arabidopsis. Nature 363:715–717PubMedCrossRefGoogle Scholar
  2. Aarts MG, Hodge R, Kalantidis K, Florack D, Wilson ZA, Mulligan BJ, Stiekema WJ, Scott R, Pereira A (1997) The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes. Plant J 12:615–623PubMedCrossRefGoogle Scholar
  3. Alexander MP (1969) Differential staining of aborted and nonaborted pollen. Stain Technol 44:117–122PubMedGoogle Scholar
  4. An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilpeoort RA (eds) Plant molecular biology manual. Kluwer, Dordrecht, pp 1–19Google Scholar
  5. Beers EP, Woffenden BJ, Zhao C (2000) Plant proteolytic enzymes: possible roles during programmed cell death. Plant Mol Biol 44:399–415PubMedCrossRefGoogle Scholar
  6. Cheng H, Qin LJ, Lee SC, Fu XD, Richards DE (2004) Gibberellin regulates Arabidopsis floral development via suppression of DELLA protein function. Development 131:1055–1064PubMedCrossRefGoogle Scholar
  7. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  8. Coupe SA, Sinclair BK, Watson LM, Heyes JA, Eason JR (2003) Identification of dehydration-responsive cysteine proteases during post-harvest senescence of broccoli florets. J Exp Bot 54:1045–1056PubMedCrossRefGoogle Scholar
  9. Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:581–586PubMedCrossRefGoogle Scholar
  10. Eason JR, Ryan DJ, Watson LM, Hedderley D, Christey MC, Braun RH, Coupe SA (2005) Suppression of the cysteine protease, aleurain, delays floret senescence in Brassica oleracea. Plant Mol Biol 57:645–657PubMedCrossRefGoogle Scholar
  11. Glover J, Grelon M, Craig S, Chaudhury A, Dennis E (1998) Cloning and characterization of MS5 from Arabidopsis: a gene critical in male meiosis. Plant J 15:345–356PubMedCrossRefGoogle Scholar
  12. Hatakeyama K, Ishiguro S, Okada K, Takasaki T, Hinata K (2003) Antisense inhibition of a nuclear gene, BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment. Mol Breed 11:325–336CrossRefGoogle Scholar
  13. Hayashi Y, Yamada K, Shimada T, Matsushima R, Nishizawa NK, Nishimura M, Hara-Nishimura I (2001) A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol 42:894–899PubMedCrossRefGoogle Scholar
  14. Hedden P, Phillips AL, Coles J, Thomas S, Appleford N, Ward D, Beale M, Lenton J (1999) Gibberellin biosynthesis: genes, regulation and genetic manipulation. RIKEN Rev 21:29–30Google Scholar
  15. Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, O’Gara F, Haas D (2000) Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol Plant Microbe Interact 13:232–237PubMedCrossRefGoogle Scholar
  16. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  17. Hird DL, Worrall D, Hodge R, Smartt S, Paul W, Scott R (1993) The anther-specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to β-1, 3-glucanases. Plant J 4:1023–1033PubMedCrossRefGoogle Scholar
  18. Huang S, Cerny RE, Qi YL, Bhat D, Aydt CM (2003) Transgenic studies on the involvement of cytokinin and gibberellin in male development. Plant Physiol 131:1270–1282PubMedCrossRefGoogle Scholar
  19. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  20. Jung KH, Han MJ, Lee YS, Kim YW, Hwang I, Kim MJ, Kim YK, Nahm BH, An G (2005) Rice Undeveloped Tapetum1 is a major regulator of early tapetum development. Plant Cell 17:2705–2722PubMedCrossRefGoogle Scholar
  21. Kawanabe T, Ariizumi T, Kawai-Yamada M, Uchimiya H, Toriyama K (2006) Abolition of the tapetum suicide program ruins microsporogenesis. Plant Cell Physiol 47:784–787PubMedCrossRefGoogle Scholar
  22. Kiba T, Aoki K, Sakakibara H, Mizuno T (2004) Arabidopsis response regulator, ARR22, ectopic expression of which results in phenotypes similar to the wol cytokinin-receptor mutant. Plant Cell Physiol 45:1063–1077PubMedCrossRefGoogle Scholar
  23. Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K (1993) Structure and expression of two genes that encode distinct drought-inducible cysteine proteinases in Arabidopsis thaliana. Gene 129:175–182PubMedCrossRefGoogle Scholar
  24. Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different temporal and spatial gene expression patterns occur during anther development. Plant Cell 2:1201–1224PubMedCrossRefGoogle Scholar
  25. Koncz C, Schell J (1986) The promoter of the TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396CrossRefGoogle Scholar
  26. Lee S, Jung KH, An G, Chung YY (2004) Isolation and characterization of a rice cysteine protease gene, OsCP1, using T-DNA gene-trap system. Plant Mol Biol 54:755–765PubMedCrossRefGoogle Scholar
  27. Lee YH, Chung KH, Kim HU, Jin YM, Kim HI, Park BS (2003) Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L. ssp. pekinensis. Plant Cell Rep 22:268–273PubMedCrossRefGoogle Scholar
  28. Li N, Zhang DS, Liu HS, Yin CS, Li XX, Liang WQ, Yuan Z, Xu B, Chu HW, Wang J, Wen TQ, Huang H, Luo D, Ma H, Zhang DB (2006) The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development. Plant Cell 18:2999–3014PubMedCrossRefGoogle Scholar
  29. Li Y, Cheng ZM, Smith WA, Ellis DR, Chen YQ, Zheng XL, Pei Y, Luo KM, Zhao DG, Yao QH, Duan H, Li Q (2004) Invasive ornamental plants: Problems, challenges, and molecular tools to neutralize their invasiveness. CRC Crit Rev Plant Sci 23:381–389CrossRefGoogle Scholar
  30. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8:457–463PubMedCrossRefGoogle Scholar
  31. Mariani C, Debeuckeleer M, Truettner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347:737–741CrossRefGoogle Scholar
  32. Matsuda N, Tsuchiya T, Kishitani S, Tanaka Y, Toriyama K (1996) Partial male sterility in transgenic tobacco carrying antisense and sense PAL cDNA under the control of a tapetum-specific promoter. Plant Cell Physiol 37:215–222Google Scholar
  33. Mok DWS, Mok MC (2001) Cytokinin metabolism and action. Annu Rev Plant Physiol Plant Mol Biol 52:89–118PubMedCrossRefGoogle Scholar
  34. 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 19:611–622PubMedCrossRefGoogle Scholar
  35. Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP, Harberd NP (1997) The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses. Genes Dev 11:3194–3205PubMedCrossRefGoogle Scholar
  36. Rastogi R, Sawhney VK (1989) In vitro development of angiosperm floral buds and organs. Plant Cell Tissue Organ Cult 16:145–174Google Scholar
  37. Roberts MR, Boyes E, Scott RJ (1995) An investigation of the role of the anther tapetum during microspore development using genetic cell ablation. Sex Plant Reprod 8:299–307CrossRefGoogle Scholar
  38. Rogers HJ, Bate N, Combe J, Sullivan J, Sweetman J, Swan C, Lonsdale DM, Twell D (2001) Functional analysis of cis-regulatory elements within the promoter of the tobacco late pollen gene g10. Plant Mol Biol 45:577–585PubMedCrossRefGoogle Scholar
  39. Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu Y, 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
  40. Sawhney VK, Shukla A (1994) Male sterility in flowering plants: are plant growth substances involved? Am J Bot 81:1640–1647CrossRefGoogle Scholar
  41. Schomburg FM, Bizzell CM, Lee DJ, Zeevaart JA, Amasino RM (2003) Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 15:151–163PubMedCrossRefGoogle Scholar
  42. Scott R, Dagless E, Hodge R, Paul W, Soufleri I, Draper J (1991a) Patterns of gene expression in developing anthers of Brassica napus. Plant Mol Biol 17:195–207PubMedCrossRefGoogle Scholar
  43. Scott R, Hodge R, Paul W, Draper J (1991b) The molecular biology of anther differentiation. Plant Sci 80:167–191CrossRefGoogle Scholar
  44. Singh DP, Jermakow AM, Swain SM (2002) Gibberellins are required for seed development and pollen tube growth in Arabidopsis. Plant Cell 14:3133–3147PubMedCrossRefGoogle Scholar
  45. Sorensen AM, Kröber S, Unte US, Huijser P, Dekker K, Saedler H (2003) The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor. Plant J 33:413–423PubMedCrossRefGoogle Scholar
  46. Stieglitz H (1977) Role of beta-1, 3-glucanase in postmeiotic microspore release. Dev Biol 57:87–97PubMedCrossRefGoogle Scholar
  47. Stieglitz H, Stern H (1973) Regulation of beta-1, 3-glucanase activity in developing anthers of Lilium. Dev Biol 34:169–173PubMedCrossRefGoogle Scholar
  48. Takada K, Ishimaru K, Kamada H, Ezura H (2006) Anther-specific expression of mutated melon ethylene receptor gene Cm-ERS1/H70A affected tapetum degeneration and pollen grain production in transgenic tobacco plants. Plant Cell Rep 25:936–941PubMedCrossRefGoogle Scholar
  49. Thomas SG, Phillips AL, Hedden P (1999) Molecular cloning and functional expression of gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc Natl Acad Sci USA 96:4698–4703PubMedCrossRefGoogle Scholar
  50. Trobacher CP, Senatore A, Greenwood JS (2006) Masterminds or minions? Cysteine proteinases in plant programmed cell death. Can J Bot 84:651–667CrossRefGoogle Scholar
  51. Tsuchiya T, Toriyama K, Yoshikawa M, Ejiri S, Hinata K (1995) Tapetum-specific expression of the gene for an endo-beta-1, 3-glucanase causes male sterility in transgenic tobacco. Plant Cell Physiol 36:487–494PubMedGoogle Scholar
  52. Twell D, Yamaguchi J, Wing R, Ushiba J, McCormick S (1991) Promoter analysis of genes that are co-ordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev 5:496–507PubMedCrossRefGoogle Scholar
  53. van der Meer IM, Stam ME, van Tunen AJ, Mol JN, Stuitje AR (1992) Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. Plant Cell 4:253–262PubMedCrossRefGoogle Scholar
  54. Varnier AL, Mazeyrat-Gourbeyre F, Sangwan RS, Clement C (2005) Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation. J Struct Biol 152:118–128PubMedCrossRefGoogle Scholar
  55. Vizcay-Barrena G, Wilson ZA (2006) Altered tapetal PCD and pollen wall development in the Arabidopsis ms1 mutant. J Exp Bot 57:2709–2717PubMedCrossRefGoogle Scholar
  56. Wan L, Xia Q, Qiu X, Selvaraj G (2002) Early stages of seed development in Brassica napus: a seed coat-specific cysteine proteinase associated with programmed cell death of the inner integument. Plant J 30:1–10PubMedCrossRefGoogle Scholar
  57. Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmulling T (2003) Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15:2532–2550PubMedCrossRefGoogle Scholar
  58. Werner T, Motyka V, Strnad M, Schmulling T (2001) Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA 98:10487–10492PubMedCrossRefGoogle Scholar
  59. Whitelaw C, Paul W, Jenkins ES, Taylor VM, Roberts JA (1999) An mRNA encoding a response regulator protein from Brassica napus is upregulated during pod development. J Exp Bot 50:335–341CrossRefGoogle Scholar
  60. Willige WC, Ghosh S, Nill C, Zourelidou M, Dohmann EMN, Maier A, Schwechheimer C (2007) The DELLA domain of GA INSENSITIVE mediates the interaction with the GA INSENSITIVE DWARF1A gibberellin receptor of Arabidopsis. Plant Cell. doi:10.1105/tpc.107.051441
  61. Worrall D, Hird DL, Hodge R, Paul W, Draper J, Scott R (1992) Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco. Plant Cell 4:759–771PubMedCrossRefGoogle Scholar
  62. Xu H, Knox RB, Taylor PE, Singh MB (1995) Bcp1, a gene required for male fertility in Arabidopsis. Proc Natl Acad Sci USA 14:2106–2110CrossRefGoogle Scholar
  63. Yadegari R, Paiva G, Laux T, Koltunow AM, Apuya N, Zimmerman JL, Fischer RL, Harada JJ, Goldberg RB (1994) Cell differentiation and morphogenesis are uncoupled in Arabidopsis raspberry embryos. Plant Cell 6:1713–1729PubMedCrossRefGoogle Scholar
  64. Yamada K, Matsushima R, Nishimura M, Hara-Nishimura I (2001) A slow maturation of a cysteine protease with a granulin domain in the vacuoles of senescing Arabidopsis leaves. Plant Physiol 127:1626–1634PubMedCrossRefGoogle Scholar
  65. Yoshida H, Itoh J, Ohmori S, Miyoshi K, Horigome A, Uchida E, Kimizu M, Matsumura Y, Kusaba M, Satoh H, Nagato Y (2007) superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice. Plant Biotechnol J 5:835–846PubMedCrossRefGoogle Scholar
  66. Zhang W, Sun Y, Timofejeva L, Chen C, Grossniklaus U, Ma H (2006) Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor. Development 133:3085–3095PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Ken-ichi Konagaya
    • 1
  • Sugihiro Ando
    • 1
  • Shinichiro Kamachi
    • 1
  • Mai Tsuda
    • 1
  • Yutaka Tabei
    • 1
  1. 1.Division of Plant ScienceNational Institute of Agrobiological SciencesIbarakiJapan

Personalised recommendations