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Tissue-specific Activation Tagging in Arabidopsis thaliana for Identification and Isolation of Genes of Agronomic Importance


Affiliations
1 ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi 110 012, India
 

Activation tagging is used to recover and clone domi-nant gain-of-function alleles and usually employs a T-DNA vector containing four tandem copies of the CaMV35S enhancer sequence outward usually at the right border. This strategy, however, is not efficient as it could result in overexpression of multiple genes or genes far away from the insertion site. Therefore, we tested constitutive (CaMV35S) and tissue-specific (TFL1 and rbcS2B) promoters for activation tagging. From an initial screening of 400 T1 plants, we identi-fied several morphological variants which include seedling mutants (single cotyledon, slow seedling growth, long ischolar_main, short ischolar_main), leaf mutants (fused leaves, excess rosettes, altered shape, hyponastic leaves, bushy rosette, altered leaf polarity), inflores-cence mutants (fasciated inflorescence, inflorescence fused with the petiole of rosette leaf) and flowering time and growth habit mutants (dwarf, tall, miniature, late flowering, sturdy stem). Comparison of different activation tagging populations for different categories of mutants revealed the pre-dominance of seedling mutants in the TFL1 promoter activation tagged popu-lation; leaf and inflorescence mutant frequencies were higher in rbcS2B promoter activation tagged popula-tion, while the flowering time and growth habit mutant frequency was higher in CaMV35S promoter activation tagged population. Flanking sequence ana-lysis of three of the mutants showed that all the mu-tants carried an insertion in the intergenic region. Segregation analysis of seedlings for kanamycin resis-tance showed that on average each mutant carried about 1.3 copies of T-DNA.

Keywords

Activation Tagging, Arabidopsis thaliana, Morphological Variants, Promoters.
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  • Kazama, Y., Hirano T., Saito, H., Liu, Y., Ohbu, S., Hayashi, Y. and Abe T., Characterization of highly efficient heavy-ion muta-genesis in Arabidopsis thaliana. BMC Plant Biol., 2011, 11, 161.

  • Sundaresan, V., Springer, P., Volpe, T., Harward, S., Jones, J. D. G., Dean, C. and Martienssen, R., Patterns of gene actions in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev., 1995, 9, 1797–1810.

  • Krysan, P. J., Young, J. C. and Sussman, M. R., T-DNA as an insertional mutagen in Arabidopsis. Plant Cell, 1999, 11, 2283–2290.

  • Arabidopsis Genome Initiative, Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 2000, 408, 796–815.

  • Bouche, N. and Bouchez, D., Arabidopsis gene knockout: pheno-types wanted. Curr. Opin. Plant Biol., 2001, 4, 111–117.

  • Neff, M. M. et al., BAS1: a gene regulating brassinosteroid levels and light responsiveness in Arabidopsis. Proc. Natl. Acad. Sci. USA, 1999, 96, 15316–15323.

  • Van der Graaff, E., Dulk-Ras, A. D., Hooykaas, P. J. and Keller, B., Activation tagging of the LEAFY PETIOLE gene affects leaf petiole development in Arabidopsis thaliana. Development, 2000, 127, 4971–4980.

  • Weigel, D. et al., Activation tagging in Arabidopsis. Plant Phys-iol., 2000, 122, 1003–1013.

  • Ichikawa, T. et al., Sequence database of 1172 T-DNA insertion sites in Arabidopsis activation-tagging lines that showed pheno-types in T1 generation. Plant J., 2003, 36, 421–429.

  • Nakazawa, M. et al., Activation tagging, a novel tool to dissect the functions of a gene family. Plant J., 2003, 34, 741–750.

  • Robinson, S. J. et al., An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches. BMC Plant Biol., 2009, 9, 101.

  • Jeong, D. H. et al., Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice. Plant J., 2006, 45, 123–132.

  • Hsing, Y. I. et al., A rice activation/knockout mutant resource for high throughput functional genomics. Plant Mol. Biol., 2007, 63, 351–364.

  • Zuo, J., Niu, Q. W., Frugis, G. and Chua, N. H., The WUSCHEL gene promotes vegetative-to-embryonic transition in Arabidopsis. Plant J., 2002, 30, 349–359.

  • Matsuhara, S., Jingu, F., Takahashi, T. and Komeda, Y., Heat-shock tagging: a simple method for expression and isolation of plant genome DNA flanked by T-DNA insertions. Plant J., 2000, 22, 79–86.

  • Grant, J. J., Chini, A., Basu, D. and Loake, G. J., Targeted activa-tion tagging of the Arabidopsis NBS-LRR gene, ADR1, conveys resistance to virulent pathogens. Mol. Plant–Microbe Interact., 2003, 16, 669–680.

  • Masaki, T., Tsukagoshi, H., Mitsui, N., Nishii, T., Hattori, T., Morikami, A. and Nakamura, K., Activation tagging of a gene for a protein with novel class of CCT-domain activates expression of a subset of sugar inducible genes in Arabidopsis thaliana. Plant J., 2005, 43, 142–152.

  • Koiwa, H., Bressan, R. A. and Hasegawa, P. M., Identification of plant stress-responsive determinants in Arabidopsis by large-scale forward genetic screens. J. Exp. Bot., 2006, 57(5), 1119–1128.

  • Bradley, D., Ratcliffe, O., Vincent, C., Carpenter, R. and Coen, E., Inflorescence commitment and architecture in Arabidopsis. Sci-ence, 1997, 275(5296), 80–83.

  • Serrano-Mislata, A., Fernandez-Nohales, P., Domenech, M. J., Hanzawa, Y., Bradley, D. and Madueno, F., Separate elements of the TERMINAL FLOWER 1 cis-regulatory region integrate path-ways to control flowering time and shoot meristem identity. Development, 2016, 143, 3315–3327.

  • Kushwah, N. S., Ahmad, I. and Ali, S., Characterization of pro-moter of Terminal Flower1 (TFL1) gene of Arabidopsis. Res. J. Biotechnol., 2014, 9(3), 35–40.

  • Bakhsh, A. and Husnain, T., Endeavours of Rubisco small subunit promoter as a tool of green tissue specific expression. Czech. J. Genet. Plant Breed., 2012, 48, 1–9.

  • Donald, R. G. and Cashmore, A. R., Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter. EMBO J., 1990, 9, 1717–1726.

  • Martinez-Hernandez, A., Lopez-Ochoa, L., Arguello-Astorga, G. and Herrera-Estrella, L., Functional properties and regulatory complexity of a minimal RBCS light-responsive unit activated by phytochrome, cryptochrome, and plastid signals. Plant Physiol., 2002, 128, 1223–1233.

  • Kushwah, N. S., Isolation, cloning and characterization of pro-moter of Rubisco small subunit 2B (rbcS2B) gene of Arabidopsis thaliana. Innov. Farm., 2016, 1(4), 119–128.

  • Sawchuk, M. G., Donner, T. J., Head, P. and Scarpella, E., Unique and overlapping expression patterns among members of photosyn-thesis-associated nuclear gene families in Arabidopsis. Plant Physiol., 2008, 148, 1908–1924.

  • Kim, J. Y., Yuan, Z. and Jackson, D., Developmental regulation and significance of KNOX protein trafficking in Arabidopsis. Development, 2003, 130, 4351–4362.

  • Jefferson, R. A., Kavanagh, T. and Bevan, M. W., GUS fusion: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 1987, 6, 3901–3907.

  • Clough, S. J. and Bent, A. F., Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J., 1998, 16, 735–743.

  • Liu, Y. G., Mitsukawa, N., Oosumi, T. and Whittier, R. F., Effi-cient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J., 1995, 8, 457–463.

  • O’Malley, R. C., Alonso, J. M., Kim, C. J., Leisse, T. J. and Eck-er, J. R., An adapter ligation-mediated PCR method for high-throughput mapping of T-DNA inserts in the Arabidopsis genome. Nature, 2007, 2(11), 2910–2917.

  • Livak, K. J. and Schmittgen, T. D., Analysis of relative gene ex-pression data using real-time quantitative PCR and the 2–CT method. Methods, 2001, 25, 402–408.

  • Gheysen, G., Van Montagu, M. and Zambryskl, P., Integration of Agrobacterium tumefaciens transfer DNA (T-DNA) involves rear-rangements of target plant DNA sequences. Proc. Natl. Acad. Sci. USA, 1987, 84(17), 6169–6173.

  • Ichikawa, T. et al., The FOX hunting system: an alternative gain-of-function gene hunting technique. Plant J., 2006, 48, 974–985.

  • McElver, J. et al., Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana. Genetics, 2001, 159(4), 1751–1763.

  • Li, Y., Rosso, M. G., Ulker, B. and Weisshaar, B., Analysis of T-DNA insertion site distribution patterns in Arabidopsis thaliana reveals special features of genes without insertions. Genomics, 2006, 87, 645–652.

  • Kay, R., Chan, A., Daly, M. and McPherson, J., Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science, 1987, 236(4806), 1299–302.


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  • Tissue-specific Activation Tagging in Arabidopsis thaliana for Identification and Isolation of Genes of Agronomic Importance

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Authors

Kushwah Neetu Singh
ICAR-National Research Centre on Plant Biotechnology, Pusa Campus, New Delhi 110 012, India

Abstract


Activation tagging is used to recover and clone domi-nant gain-of-function alleles and usually employs a T-DNA vector containing four tandem copies of the CaMV35S enhancer sequence outward usually at the right border. This strategy, however, is not efficient as it could result in overexpression of multiple genes or genes far away from the insertion site. Therefore, we tested constitutive (CaMV35S) and tissue-specific (TFL1 and rbcS2B) promoters for activation tagging. From an initial screening of 400 T1 plants, we identi-fied several morphological variants which include seedling mutants (single cotyledon, slow seedling growth, long ischolar_main, short ischolar_main), leaf mutants (fused leaves, excess rosettes, altered shape, hyponastic leaves, bushy rosette, altered leaf polarity), inflores-cence mutants (fasciated inflorescence, inflorescence fused with the petiole of rosette leaf) and flowering time and growth habit mutants (dwarf, tall, miniature, late flowering, sturdy stem). Comparison of different activation tagging populations for different categories of mutants revealed the pre-dominance of seedling mutants in the TFL1 promoter activation tagged popu-lation; leaf and inflorescence mutant frequencies were higher in rbcS2B promoter activation tagged popula-tion, while the flowering time and growth habit mutant frequency was higher in CaMV35S promoter activation tagged population. Flanking sequence ana-lysis of three of the mutants showed that all the mu-tants carried an insertion in the intergenic region. Segregation analysis of seedlings for kanamycin resis-tance showed that on average each mutant carried about 1.3 copies of T-DNA.

Keywords


Activation Tagging, Arabidopsis thaliana, Morphological Variants, Promoters.

References





DOI: https://doi.org/10.18520/cs%2Fv117%2Fi4%2F627-637