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Transient overexpression of E2Fb triggers cell divisions in pavement cells of Nicotiana benthamiana leaves

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Abstract

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Agrobacterium-mediated transient overexpression of E2Fb triggers new cell divisions in pavement cells of Nicotiana benthamiana leaves.

Abstract

Transient transformation in Nicotiana benthamiana enables the study of multiple biological processes in a simple and fast manner. Here, we describe that, upon A. tumefaciens-mediated transient overexpression of the cell cycle regulator E2Fb from either Arabidopsis thaliana or N. benthamiana, cell divisions occur in epidermal pavement cells in N. benthamiana leaves, following a sequence of events that encompasses the nucleus taking a central position and being surrounded by chloroplasts, nuclear division, and formation of a new wall that divides the initial cell in two. Our results indicate that transient expression in N. benthamiana can be used to study cell division in plants, from DNA replication to cell wall formation, in a simple, controlled, and rapid manner.

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References

  • Chandran D, Inada N, Hather G, Kleindt CK, Wildermuth MC (2010) Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators. PNAS 107(1):460–465

    Article  CAS  Google Scholar 

  • Chandran D, Rickert J, Cherk C, Dotson BR, Wildermuth MC (2013) Host cell ploidy underlying the fungal feeding site is a determinant of powdery mildey growth and reproduction. MPMI 26(5):537–545

    Article  CAS  Google Scholar 

  • De Veylder L, Beeckman T, Beemster GT, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A, Engler G et al (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa–DPa transcription factor. EMBO J 21:1360–1368

    Article  Google Scholar 

  • del Pozo JC, Diaz-Trivino S, Cisneros N, Gutierrez C (2006) The balance between cell division and endoreplication depends on E2FC–DPB, transcription factors regulated by the ubiquitin-SCFSKP2A pathway in Arabidopsis. Plant Cell 18:2224–2235

    Article  Google Scholar 

  • Desvoyes B, Ramirez-Parra E, Xie Q, Chua NH, Gutierrez C (2006) Cell type-specific role of the retinoblastoma/E2F pathway during Arabidopsis leaf development. Plant Phys 140:67–80

    Article  CAS  Google Scholar 

  • Donnelly PM, Bonetta D, Tsukaya H, Dengler RE, Dengler NG (1999) Cell cycling and cell enlargement in developing leaves of Arabidopsis. Dev Biol 215:407–419

    Article  CAS  Google Scholar 

  • Goodin M, Chakrabarty R, Yelton S (2008) Membrane and protein dynamics in virus-infected plant cells. Methods Mol Biol 451:377–393

    Article  CAS  Google Scholar 

  • Heckmann S, Lermontova I, Berckmans B, De Veylder L, Baumlein H, Schubert I (2011) The E2F transcription factor family regulates CENH3 expression in Arabidopsis thaliana. Plant J 68:646–656

    Article  CAS  Google Scholar 

  • Hemerly A, Engler Jde A, Bergounioux C, Van Montagu M, Engler G, Inze D, Ferreira P (1995) Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development. EMBO J 14:3925–3936

    Article  CAS  Google Scholar 

  • Henriques R, Magyar Z, Bogre L (2013) S6K1 and E2FB are in mutually antagonistic regulatory links controlling cell growth and proliferation in Arabidopsis. Plant Signal Behav 8:e24367

    Article  Google Scholar 

  • Inze D (2005) Green light for the cell cycle. EMBO J 24:657–662

    Article  CAS  Google Scholar 

  • Kosugi S, Ohashi Y (2002) Interaction of the Arabidopsis E2F and DP proteins confers their concomitant nuclear translocation and transactivation. Plant Phys 128:833–843

    Article  CAS  Google Scholar 

  • Liu CJ, Dixon RA (2001) Elicitor-induced association of isoflavone O-methyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis. Plant Cell 13:2643–2658

    Article  CAS  Google Scholar 

  • Lu Q, Tang X, Tian G, Wang F, Liu K, Nguyen V, Kohalmi SE, Keller WA, Tsang EW, Harada JJ, Rothstein SJ, Cui Y (2010) Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J 61(2):259–270. https://doi.org/10.1111/j.1365-313X.2009.04048.x

    Article  CAS  PubMed  Google Scholar 

  • Magyar Z, De Veylder L, Atanassova A, Bako L, Inze D, Bogre L (2005) The role of the Arabidopsis E2FB transcription factor in regulating auxin-dependent cell division. Plant Cell 17:2527–2541

    Article  CAS  Google Scholar 

  • Mariconti L, Pellegrini B, Cantoni R, Stevens R, Bergounioux C, Cella R, Albani D (2002) The E2F family of transcription factors from Arabidopsis thaliana. Novel and conserved components of the retinoblastoma/E2F pathway in plants. JBC 277:9911–9919

    Article  CAS  Google Scholar 

  • Mason G, Caciagli P, Accotto GP, Noris E (2008) Real-time PCR for the quantitation of tomato yellow leaf curl Sardinia virus in tomato plants and in Bemisia tabaci. J Virol Methods 147:282–289

    Article  CAS  Google Scholar 

  • Menges M, de Jager SM, Gruissem W, Murray JA (2005) Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, reveals multiple and highly specific profiles of expression and provides a coherent model for plant cell cycle control. Plant J 41:546–566

    Article  CAS  Google Scholar 

  • Nakagawa T et al (2007a) Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J Biosci Bioeng 104(1):34–41

    Article  CAS  Google Scholar 

  • Nakagawa T et al (2007b) Improved gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci Biotechnol Biochem 71(8):2095–2100

    Article  CAS  Google Scholar 

  • Otero S, Desvoyes B, Gutierrez C (2016) Histone H3 dynamics reveals domains with distinct proliferation potential in the Arabidopsis root. Plant Cell 28(6):1361–1371

    Article  CAS  Google Scholar 

  • Vandepoele K, Raes J, De Veylder L, Rouze P, Rombauts S, Inze D (2002) Genome-wide analysis of core cell cycle genes in Arabidopsis. Plant Cell 14:903–916

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Shingo Nagawa and Li Tan at the Cell Biology Core Facility at the Shanghai Center for Plant Stress Biology for useful advice and excellent technical assistance.

Funding

Research in the RL-D’s lab is funded by the Shanghai Center for Plant Stress Biology of the Chinese Academy of Sciences and the 100 Talent program of the Chinese Academy of Sciences. TJ-G was sponsored by a CAS-TWAS President’s Fellowship for International Ph.D. students.

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Authors and Affiliations

  1. Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, 201602, China

    Tamara Jiménez-Gόngora, Huang Tan & Rosa Lozano-Durán

  2. University of the Chinese Academy of Sciences, Beijing, 100049, China

    Tamara Jiménez-Gόngora & Huang Tan

Authors
  1. Tamara Jiménez-Gόngora
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  2. Huang Tan
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  3. Rosa Lozano-Durán
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Contributions

RL-D and TJ-G conceived the project; TJ-G and HT performed experiments and analyzed data; RL-D wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Rosa Lozano-Durán.

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Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Communicated by Da-Bing Zhang.

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Electronic supplementary material

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Supplementary material 1 (DOCX 31 kb).

Supplementary material 2 (WMV 9791 kb) Supplementary video 1. A dividing epidermal pavement cell of N. benthamiana transiently co-expressing YFP-AtE2Fb and the nuclear marker AtHTR5-CFP.

299_2019_2457_MOESM3_ESM.jpg

Supplementary material 3 (JPEG 69 kb) Figure S1. Transient overexpression of AtE2Fb, but not AtDPa or AtDPb, induces cell divisions in epidermal pavement cells of Nicotiana benthamiana leaves. (A) Nuclear localization of GFP-AtDPa, GFP-AtDPb, and GFP-AtE2Fb in pavement cells of N. benthamiana leaves upon transient overexpression. Scale bar = 25 µm. (B) Western blot analysis of protein accumulation in samples expressing GFP-AtDPa, GFP-AtDPb, and GFP-AtE2Fb.

299_2019_2457_MOESM4_ESM.jpg

Supplementary material 4 (JPEG 82 kb) Figure S2. Analysis of ploidy levels in pavement cells of Nicotiana benthamiana leaves. (A) Example of maximum projection image of pavement cells of N. benthamiana after DAPI staining. Arrowheads point at nuclei of stomatal guard cells. Scale bar = 100 µm. (B) 3D view of selected nuclei analyzed by Imaris (V7.7, Bitplane, Inc., MN). Arrowheads point at nuclei of stomatal guard cells. Scale bar = 100 µm. (C) DNA ploidy distribution (percentage) in pavement cells of N. benthamiana. n = 147 nuclei. Ploidy levels were estimated based on DAPI fluorescence values normalized to those in stomatal guard cells (2n), as described in Chandran et al., (2010, 2013).

299_2019_2457_MOESM5_ESM.jpg

Supplementary material 5 (JPEG 34 kb) Figure S3. Relative expression levels of NbDP in pavement cells of N. benthamiana transiently overexpressing Arabidopsis GFP-DPa, GFP-DPb, or GFP-E2Fb. Transcript levels were normalized to ITS and are shown relative to those in the GFP control. Data are the mean fold difference ± SD of three biological replicates with three technical repeats each.

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Jiménez-Gόngora, T., Tan, H. & Lozano-Durán, R. Transient overexpression of E2Fb triggers cell divisions in pavement cells of Nicotiana benthamiana leaves. Plant Cell Rep 38, 1465–1471 (2019). https://doi.org/10.1007/s00299-019-02457-3

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