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

  • Tamara Jiménez-Gόngora
  • Huang Tan
  • Rosa Lozano-DuránEmail author
Original Article

Abstract

Key message

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.

Keywords

Nicotiana bentamiana E2Fb Cell division Pavement cell Transient expression 

Notes

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.

Author contribution statement

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.

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.

Compliance with ethical standards

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.

Supplementary material

299_2019_2457_MOESM1_ESM.docx (31 kb)
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 (69 kb)
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 (82 kb)
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 (35 kb)
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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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant SciencesChinese Academy of SciencesShanghaiChina
  2. 2.University of the Chinese Academy of SciencesBeijingChina

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