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Protocol for screening and expression studies of T-DNA and tagging-based insertional knox mutants in Arabidopsis thaliana

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Abstract

KNOTTED1-like homeobox (KNOX) genes serve important roles in meristem function and many developmental processes in all higher plants. In Arabidopsis, studies of KNOX genes especially among members of class II KNOX genes remain limited and functional data are largely lacking. In the present study, we established a reproducible protocol that is important for genetic studies of KNOX genes using Arabidopsis insertional mutants. This protocol contains a reproducible and serial procedure containing detailed and step-by-step laboratory and field works covering all experiment steps from the screening of homozygous mutant lines to the KNOX expression analysis using qRT-PCR in a single paper. The troubleshooting and challenges that might occur are also presented and discussed. T-DNA insertion mutants for all Arabidopsis KNOX genes (except for knat4) were isolated based on kanamycin screening, phenotype selection, and PCR genotyping. Surprisingly, the insertions resulted in strong repression of the respective KNOX genes. However, no gene suppression was observed for the positively selected knat5 mutant. Moreover, qRT-PCR was effective for transcript analysis among the knox mutant samples. The use of different relative expression quantification produces a similar indication of expression level. Overall, the proposed procedure is highly effective for expression studies of KNOX genes in Arabidopsis mutants and will serve as a fundamental work protocol to open opportunities for genetic studies of genes involving insertional mutants in Arabidopsis.

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References

  • Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, 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 DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657

    Article  PubMed  Google Scholar 

  • Barton MK, Poethig SR (1993) Formation of the shoot apical meristem in Arabidopsis thaliana: an analysis of development in the wild type and in the shoot meristemless mutant. Development 119:823–831

    Article  Google Scholar 

  • Belles-Boix E, Hamant O, Witiak SM, Morin H, Traas J, Pautot V (2006) KNAT6: An Arabidopsis homeobox gene involved in meristem activity and organ separation. Plant Cell 18:1900–1907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bolle C, Schneider A, Leister D (2011) Perspectives on systematic analyses of gene function in Arabidopsis thaliana: new tools, topics and trends. Curr Genomics 12:1–14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chuck G, Lincoln C, Hake S (1996) KNAT1 induces lobed leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell 8:1277–1289

    CAS  PubMed  PubMed Central  Google Scholar 

  • Clark SE, Jacobsen SE, Levin JZ, Meyerowitz EM (1996) The CLAVATA and SHOOT MERISTEMLESS loci competitively regulate meristem activity in Arabidopsis. Development 122:1567–1575

    Article  CAS  PubMed  Google Scholar 

  • Dean G, Casson S, Lindsey K (2004) KNAT6 gene of Arabidopsis is expressed in roots and is required for correct lateral root formation. Plant Mol Biol 54(1):71–84

    Article  CAS  PubMed  Google Scholar 

  • Edwards K, Jonstone C, Thomson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acid Res 19:1349–1350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feldmann KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum. Plant J 1:71–82

    Article  CAS  Google Scholar 

  • Francis KE, Spiker S (2005) Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations. Plant J 41:464–477

    Article  CAS  PubMed  Google Scholar 

  • Geniza M, Jaiswal P (2017) Tools for building de novo transcriptome assembly. Curr Plant Biol 11–12:41–45

    Article  Google Scholar 

  • Hake S, Smith HMS, Holtan H, Magnani E, Mele G, Ramirez J (2004) The role of KNOX genes in plant development. Ann Rev Cell Dev Biol 20:125–151

    Article  CAS  Google Scholar 

  • Hay A, Tsiantis M (2010) KNOX genes: versatile regulators of plant development and diversity. Development 137:3153–3165

    Article  CAS  PubMed  Google Scholar 

  • Kim SI, Veena GSB (2007) Genome-wide analysis of Agrobacterium T-DNA integration sites in the Arabidopsis genome generated under non-selective conditions. Plant J 51:779–791

    Article  CAS  PubMed  Google Scholar 

  • Koncz C, Schell J, Rkdei GP (1992) T-DNA transformation and insertion mutagenesis. In: Koncz C, Chua N-H, Schell J (eds) Methods in Arabidopsis research. World Scientific, Singapore, pp 224–273

    Chapter  Google Scholar 

  • Krysan PJ, Young JC, Sussman MR (1999) T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 11:2283–2290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li E, Bhargava A, Qiang W, Friedmann MC, Forneris N, Savidge RA, Johnson LA, Mansfield SD, Ellis BE, Douglas CJ (2012) The Class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. New Phytol 194(1):102–115

    Article  CAS  PubMed  Google Scholar 

  • Long JA, Moan EI, Medford JI, Barton MK (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature 379:66–69

    Article  CAS  PubMed  Google Scholar 

  • Mele G, Ori N, Sato Y, Hake S (2003) The knotted1-like homeobox gene BREVIPEDICELLUS regulates cell differentiation by modulating metabolic pathways. Genes Dev 19:412–412

    Google Scholar 

  • O’Malley RC, Barragan CC, Ecker JR (2015) A user’s guide to the Arabidopsis T-DNA insertional mutant collections. Methods Mol Biol 1284:323–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):2001–2007

    Article  Google Scholar 

  • Qin W, Yin Q, Chen J, Zhao X, Yue F, He J, Yang L, Liu L, Zeng Q, Lu F, Mitsuda N, Ohme-Takagi M, Wu A (2020) The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis. J Exp Bot 71(18):5469–5483

    Article  CAS  PubMed  Google Scholar 

  • Rhee SY, Mutwil M (2014) Towards revealing the functions of all genes in plants. Trends Plant Sci 19:212–221

    Article  CAS  PubMed  Google Scholar 

  • Scofield S, Murray JAH (2006) KNOX gene function in plant stem cell niches. Plant Mol Biol 60:929–946

    Article  CAS  PubMed  Google Scholar 

  • Scofield S, Dewitte W, Nieuwland J, Murray JAH (2013) The Arabidopsis homeobox gene SHOOT MERISTEMLESS has cellular and meristem-organisational roles with differential requirements for cytokinin and CYCD3 activity. Plant J 75:53–66

    Article  CAS  PubMed  Google Scholar 

  • The Arabidopsis Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

    Article  Google Scholar 

  • Truernit E, Haseloff J (2007) A Role for KNAT class II genes in root development. Plant Signal Behav 2(1):10–12

    Article  PubMed  PubMed Central  Google Scholar 

  • Venglat SP, Dumonceaux T, Rozwadowski K, Parnell L, Babic V, Keller W, Martienssen R, Selvaraj G, Datla R (2002) The homeobox gene BREVIPEDICELLUS is a key regulator of inflorescence architecture in Arabidopsis. Proc Nat Acad Sci USA 99:74730–74735

    Article  Google Scholar 

  • Weigel D, Glazebrook J (2006) Setting up Arabidopsis crosses. CSH Protocols 5

  • Woerlen N, Allam G, Popescu A, Corrigan L, Pautot V, Hepworth SR (2017) Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein. Planta 245(6):1079–1090

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The author would like to express a sincere gratitude to Dr. Urs Fischer and Prof. Andrea Polle (Department of Forest Botany and Tree Physiology, University of Goettingen) for their significant advices in experiment design and analysis. The author also wishes to thank Angela Hay (University of Oxford, UK) for providing knat1bp-9 seed stocks.

Funding

This work was funded by the Deutsche Forschungsgemeinschaft ‘Pappelgruppe’ (Andrea.Polle),

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  1. Widi Sunaryo

    Present address: Department of Agroecotechnology, Faculty of Agriculture, Mulawarman University, Jl. Pasir Balengkong, Kampus Gunung Kelua, Samarinda, East Kalimantan, 75123, Indonesia

Authors and Affiliations

  1. Department of Forest Botany and Tree Physiology, University of Göttingen, E37077, Göttingen, Germany

    Widi Sunaryo

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  1. Widi Sunaryo
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Contributions

The author designed, performed, and analyzed the experiments, as well as wrote the manuscript. The author got valuable guidances and advises from Dr. Urs Fischer (Department of Forest Botany and Tree Physiology, University of Goettingen) in research design, material selection, data analysis and interpretation. Author also got significant advices and financial support from Prof. Andrea Polle (The Head of Department of Forest Botany and Tree Physiology, University of Goettingen) through the poplar research group “Deutsche Forschungsgemeinschaft ‘Pappelgruppe’.

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Correspondence to Widi Sunaryo.

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The author declares no competing financial interests.

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Sunaryo, W. Protocol for screening and expression studies of T-DNA and tagging-based insertional knox mutants in Arabidopsis thaliana. 3 Biotech 11, 332 (2021). https://doi.org/10.1007/s13205-021-02868-8

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