Abstract
Gene containment technologies that prevent transgene dispersal through pollen, fruit and seed are in immediate demand to address concerns of gene flow from transgenic crops into wild species or close relatives. In this study, we isolated the enhancer element of Arabidopsis AGAMOUS that drives gene expression specifically in stamens and carpels. By fusing this AG enhancer to a minimal 35S promoter fragment, two tissue-specific promoters, fAGIP and rAGIP in forward and reverse orientations, respectively, were created and fused to the GUS reporter. Transgenic Arabidopsis plants harboring either fAGIP::GUS or rAGIP::GUS displayed similar GUS expression specifically in carpel and stamen tissues and their primordial cells. To test their utility for engineering sterility, the promoters were fused to the Diphtheria toxin A (DT-A) gene coding for a ribosome inactivating protein as well as the Barnase gene coding for an extracellular ribonuclease, and tested for tissue-specific ablation. Over 89% of AGIP::DT-A and 68% of AGIP::Barnase transgenic plants displayed specific and precise ablation of stamens and carpels and are completely sterile. These transgenic plants showed normal vegetative development with prolonged vegetative growth. To evaluate the stability of the sterile phenotype, 16 AGIP::DT-A lines underwent two consecutive cutback generations and showed no reversion of the floral phenotype. This study demonstrates a simple, precise and efficient approach to achieve absolute sterility through irreversible ablation of both male and female floral organs. This approach should have a practical application for transgene containment in ornamental, landscaping, and woody species, whose seeds and fruits are of no economic value.
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References
Bao X, Franks RG, Levin JZ, Liu Z (2004) Repression of AGAMOUS by BELLRINGER in floral and inflorescence meristems. Plant Cell 16:1478–1489
Beals TP, Goldberg RB (1997) A novel cell ablation strategy blocks tobacco anther dehiscence. Plant Cell 9:1527–1545
Benedito VA, Visser PB, van Tuyl JM, Angenent GC, de Vries SC, Krens FA (2004) Ectopic expression of LLAG1, an AGAMOUS homologue from lily (Lilium longiflorum Thunb.) causes floral homeotic modifications in Arabidopsis. J Exp Bot 55:1391–1399
Blazquez MA, Soowal LN, Lee I, Weigel D (1997) LEAFY expression and flower initiation in Arabidopsis. Development 124:3835–3844
Block M, Debrouwer D (1993) Engineered fertility control in transgenic Brassica napus L: histochemical analysis of anther development. Planta 189:218–225
Block M, Debrouwer D, Moens T (1997) The development of a nuclear male sterility system in wheat: Expression of the barnase gene under the control of tapetum specific promoters. Theor Appl Genet 95:125–131
Brunner AM, Rottmann WH, Sheppard LA, Krutovskii K, DiFazio SP, Leonardi S, Strauss SH (2000) Structure and expression of duplicate AGAMOUS orthologues in poplar. Plant Mol Biol 44:619–634
Burgess DG, Ralston EJ, Hanson WG, Heckert M, Ho M, Jeng T, Palys JM, Tang K, Gutterson N (2002) A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants. Plant J 31:113–125
Busch MA, Bomblies K, Weigel D (1999) Activation of a floral homeotic gene in Arabidopsis. Science 285:585–587
Clough SJ, Bent AF (1998) Floral Dip: a simple method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
Dale PJ, Clarke B, Fontes EMG (2002) Potential for the environmental impact of transgenic crops. Nature Biotechnol 20:567–574
Day CD, Galgoci BFC, Irish VF (1995) Genetic ablation of petal and stamen primordial to elucidate interactions during plant development. Development 121:2887–2895
Deyholos MK, Sieburth LE (2000) Separable whole-specific expression and negative regulation by enhancer elements within the AGAMOUS second intron. Plant Cell 12:1799–1810
Drews GN, Bowman JL, Meyerowitz EM (1991) Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product. Cell 65:991–1002
Ellstrand NC (2001) When transgenes wander, should we worry? Plant Physiol 125:1543–1545
Ellstrand NC (2003) Going to “Great Length” to prevent the escape of genes that produce specialty chemicals. Plant Physiol 132:1770–1774
Ellstrand NC, Prentice HC, Hancock JF (1999) Gene flow and introgression from domesticated plants into their wild relatives. Annu Rev Ecol Syst 24:217–242
Gleba Y, Marillonnet S, Klimyuk V (2004) Design of safe and biologically contained transgenic plants: tools and technologies for controlled transgene flow and expression. Biotechnol Genet Eng Rev 21:325–367
Hall L, Topinka K, Huffman J, Davis L, Allen A (2000) Pollen flow between herbicide-resistant Brassica napus is the cause of multiple-resistant B. napus volunteers. Weed Sci 48:688–694
Hong RL, Hamaguchi L, Busch MA,Weigel D (2003) Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. Plant Cell 15:1296–1309
Kandasamy MK, Thorsness MK, Rundle SJ, Goldberg ML, Nasrallah JB, Nasrallah ME (1993) Ablation of papillar cell function in Brassica flowers results in the loss of stigma receptivity to pollination. Plant Cell 5:263–275
Kater MM, Colombo L, Franken J, Busscher M, Masiero S, Campagne MMVL, Angenent GC (1998) Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate. Plant Cell 10:171–182
Kempin SA, Mandel MA, Yanofsky MF (1993) Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1. Plant Phyiosl 103:1041–1046
Kobayashi K, Munemura I, Hinata K, Yamamura S (2006) Bisexual sterility conferred by the differential expression of Barnase and Barstar: a simple and efficient method of transgene containment. Plant Cell Rep 25:1347–1354
Kramer EM, Alejandra J, Stilio VSD (2004) Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in angiosperms. Genetics 166:1011–1023
Lannenpaa M, Hassinen M, Ranki A, Holtta-Vuori M, Lemmetyinen J, Keinonen K, Sopanen T (2005) Prevention of flower development in birth and other plants using a BpFULL1::BARNASE construct. Plant Cell Rep 24:69–78
Lemmetyinen J, Pennanen T, Lannenpaa M, Sopanen T (2001) Prevention of flower formation in dicotyledons. Mol Breed 7:341–350
Lemmetyinen J, Hassinen M, Elo A, Porali I, Keinonen K, Makela H, Sopanen T (2004a) Functional characterization of SEPALLASTA3 and AGAMOUS orthologues in silver birch. Physiol Plant 121:149–162
Lemmetyinen J, Keinonen K, Sopanen T (2004b) Prevention of the flowering of a tree, silver birch. Mol Breed 13:243–249
Li Y, Chang Z, Smith WA, Ellis DR, Chen Y, Zheng X, Pei Y, Luo K, Zhao D, Yao Q, Duan H, Li Q (2004) Invasive ornamental plants: problems, challenges, and molecular tools to neutralize their invasiveness. Crit Rev Plant Sci 23:381–389
Lohmann JU, Hong RL, Hobe M, Busch MA, Parcy F, Simon R, Weigel D (2001) A molecular link between stem cell regulation and floral patterning in Arabidopsis. Cell 105:793–803
Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Steward CN, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor”: fused LoxP-FRT recognition sequences dramatically improve the efficiency of FLP or Cre recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–274
Mariani C, DeBeuckeleer M, Trueltner J, Leemans J, Goldberg RB (1990) Induction of male sterility in plants by a chimeric ribonuclease gene. Nature 347:737–741
Mariani C, Gossele V, Beuckeleer MD, Block MD, Goldburg RB, Greef WD, Leemans J (1992) A chimaeric ribonuclease-inhibitor gene restores fertility to male sterile plants. Nature 357:384–387
Mikkelsen TR, Andersen B, Jorgensen RB (1996) The risk of crop transgene spread. Nature 380:31
Nilsson O, Wu E, Wolfe DS, Weigel D (1998) Genetic ablation of flowers in transgenic Arabidopsis. Plant J 15:799–804
Pnueli L, Hareven D, Rounsley S, Yanofsky MF, Lifschitz E (1994) Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants. Plant Cell 6:163–173
Rutledge R, Regan S, Nicolas O, Fobert P, Cote C, Bosnich W, Kauffeldt C, Sunohara G, Seguin A, Steward D (1998) Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis. Plant J 15:625–634
Sieburth LE, Meyerowitz E (1997) Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355–365
Skinner JS, Meilan R, Ma C, Strauss SH (2003) The Populus PTD promoter imparts floral-predominant expression and enables high levels of floral-organ ablation in Populus, Nicotiana and Arabidopsis. Mol Breed 12:119–132
Snow AA (2002) Transgenic crops why gene flow matters. Nat Biotechnol 20:542–542
Snow AA, Palma PM (1997) Commercialization of transgenic plants: Potential ecological risks. Bioscience 47:86–96
Thorsness MK, Kandasamy MK, Nasrallah ME, Nasrallah JB (1993) Genetic ablation of floral cells in Arabidopsis. Plant Cell 5:253–261
Wei H, Meilan R, Brunner AM, Skinner JS, Ma K, Gandhi HT, Strauss SH (2007) Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene. Mol Breed 19:69–85
Yanofsky M F, Ma H, Bowman J L, Drews G N, Feldmann K A, Meyerowitz E M (1990) The protein encoded by the Arabidopsis homeotic gene AGAMOUS resembles transcription factors. Nature 346:35–39
Zahn LM, Leebens-Mack JH, Arrington JM, Hu Y, Landherr L, dePamphilis CW, Becker A, Theissen G, Ma H (2006) Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events. Evol Dev 8:30–45
Zhang P, Tan HTW, Pwee K-H, Humar PP (2004) Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms. Plant J 37:566–577
Acknowledgments
We thank Mr. Dennis Bennett for technical assistance. This work was supported by a USDA CSREES BRAG grant to Zongrang Liu.
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Communicated by K. Kamo.
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Liu, Z., Liu, Z. The second intron of AGAMOUS drives carpel- and stamen-specific expression sufficient to induce complete sterility in Arabidopsis . Plant Cell Rep 27, 855–863 (2008). https://doi.org/10.1007/s00299-008-0511-3
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DOI: https://doi.org/10.1007/s00299-008-0511-3