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Live single-cell transcriptional dynamics via RNA labelling during the phosphate response in plants

Nature Plants volume 7, pages 1050–1064 (2021)Cite this article

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Abstract

Plants are constantly adapting to ambient fluctuations through spatial and temporal transcriptional responses. Here, we implemented the latest-generation RNA imaging system and combined it with microfluidics to visualize transcriptional regulation in living Arabidopsis plants. This enabled quantitative measurements of the transcriptional activity of single loci in single cells, in real time and under changing environmental conditions. Using phosphate-responsive genes as a model, we found that active genes displayed high transcription initiation rates (one initiation event every ~3 s) and frequently clustered together in endoreplicated cells. We observed gene bursting and large allelic differences in single cells, revealing that at steady state, intrinsic noise dominated extrinsic variations. Moreover, we established that transcriptional repression triggered in roots by phosphate, a crucial macronutrient limiting plant development, occurred with unexpectedly fast kinetics (on the order of minutes) and striking heterogeneity between neighbouring cells. Access to single-cell RNA polymerase II dynamics in live plants will benefit future studies of signalling processes.

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Fig. 1: Identification of fast responsive transcripts regulated at the transcriptional level by phosphate resupply.
Fig. 2: Validation of pSPX1::MS2×128 transgenic plants.
Fig. 3: Imaging of SPX1 transcription in fixed plant tissues reveals allelic differences in the root cap and polyploid expression in mature tissue.
Fig. 4: Quantitation of SPX1 transcription in fixed cells gives insights into transcription dynamics, ploidy and intrinsic versus extrinsic noise.
Fig. 5: Combining microfluidic and MS2 technology reveals fast transcriptional repression triggered by Pi supply in pSPX1::MS2×128 transgenic plants.
Fig. 6: The SPX1 promoter generates bursts of activity in root cap cells grown at steady state in the absence of phosphate.
Fig. 7: Analysis of transcription site activity following Pi supply in pSPX1::MS2×128 S line transgenic plants.

Data availability

The genetic constructs, lines and datasets generated in the current study are available from the corresponding author upon request.

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Acknowledgements

S.H. was supported by a PhD fellowship from the CEA and PACA region, the ANR Reglisse 13-ADAP-008 fellowship and the CEA DRF impulsion programme, and the FOSSI project supported E.M., L.C., L.N., M.-C.T. and P.D. Additional grant support was received by H.J. from CEA-Enhanced Eurotalent and ANR PhlowZ 19-CE-13-0007. We thank the Heliobiotech platform for access to their RT–qPCR machine. We thank E. Basyuk for her help with the MS2 plasmids, and we thank L. Laplaze and G. Desbrosses for providing access to the growth chambers of IRD and Montpellier University. We thank O. Radulescu for his help with calculating total, extrinsic and intrinsic noise for an undefined number of alleles; T. Desnos and C. Mercier for their assistance on figure drawing; and S. Kanno and H. Garcia for critical reading of the manuscript. We thank J. Escudier for the synthesis of the SPX1 set of fluorescent probes. We acknowledge the MRI imaging facility (belonging to the National Infrastructure France-BioImaging supported by the French National Research Agency, ANR-10-INBS-04) and the ZoOM platform (supported by the Région Provence Alpes Côte d’Azur, the Conseil General of Bouches du Rhône, the French Ministry of Research, the Centre National de la Recherche Scientifique and the Commissariat à l’Energie Atomique et aux Energies Alternatives).

Author information

Author notes

  1. These authors contributed equally: Sahar Hani, Laura Cuyas, Pascale David.

Authors and Affiliations

  1. Aix Marseille Univ, CEA, CNRS, BIAM, UMR7265, SAVE (Signalisation pour l’Adaptation des Végétaux à leur Environnement), Saint-Paul lez Durance, France

    Sahar Hani, Laura Cuyas, Pascale David, Marie-Christine Thibaud, Nathalie Pochon, Hélène Javot & Laurent Nussaume

  2. Agroinnovation International-TIMAC AGRO, Groupe Roullier, Saint-Malo, France

    Laura Cuyas

  3. Department of Animal, Plant and Soil Sciences, Australian Research Council Centre of Excellence in Plant Energy Biology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia

    David Secco & James Whelan

  4. UMR5096 CNRS/Université de Perpignan, Laboratoire Génome et Développement des Plantes, Perpignan, France

    Rémy Merret

  5. Unité Imagerie et Modélisation, Institut Pasteur and CNRS UMR 3691, Paris, France

    Florian Mueller

  6. MRI, BioCampus Montpellier, CRBM, Univ. Montpellier, CNRS, Montpellier, France

    Orestis Faklaris

  7. UMR 5168 CNRS-CEA-INRA-Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire et Végétale, iRIG, CEA-Grenoble, Grenoble, France

    Eric Maréchal

  8. Institut de Génétique Moléculaire de Montpellier, Univ. Montpellier, CNRS, Montpellier, France

    Edouard Bertrand

  9. Institut de Génétique Humaine, Univ. Montpellier, CNRS, Montpellier, France

    Edouard Bertrand

  10. Equipe labélisée Ligue Nationale Contre le Cancer, Montpellier, France

    Edouard Bertrand

Authors
  1. Sahar Hani
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Contributions

E.B. provided the MS2 and MCP original constructs, and L.N. conceived the experiments. L.C., S.H. and P.D. performed all the experiments under the supervision of L.N. for the physiological part and E.B. for cell biology. The RNA-seq data were produced by D.S. and J.W. and analysed by L.N., L.C. M.-C.T. and E.M. The luminescence experiments were performed by N.P. under the supervision of H.J. H.J. also implemented the microfluidic technique in the SAVE team. R.M. performed the experiments for cap and polyA tail detection, F.M. provided assistance for image analysis and computation and O.F. took part in the spinning disk and mosaic acquisition experiments. The manuscript was written by L.N. and E.B. with help from S.H., P.D. and L.C.

Corresponding authors

Correspondence to Edouard Bertrand or Laurent Nussaume.

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Peer review information Nature Plants thanks Zhicheng Dong and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–12 with legends and Tables 1–3 with legends.

Reporting Summary

Supplementary Video 1

Bursting activity of the pSPX1::MS2×128 reporter. Video of root cap cells of the Arabidopsis S line expressing pSPX1::MS2×128 and MCP–GFP and continuously grown without Pi. MIPs (xy and xz) are from a time-lapse video recorded in 3D (44 z planes). Time (in min) is indicated.

Supplementary Video 2

Putative release of single RNAs in the nucleoplasm when promoter activity stochastically turns off when a burst ends. Video of root cap cells of the Arabidopsis S line expressing pSPX1::MS2×128 and MCP–GFP and continuously grown without Pi. MIPs (xy and xz) are from a time-lapse video recorded in 3D (44 z planes). Time (in min) is indicated.

Supplementary Video 3

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Video of root cells of the Arabidopsis S line transformed with pSPX1::MS2×128 and MCP–GFP, after receiving a Pi-rich solution at time t = 0 min. MIP from a time-lapse video recorded in 3D (200 z planes). Acquisitions lasted 39 min.

Supplementary Video 4

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Zoom on a few cells cropped from Supplementary Video 3.

Supplementary Video 5

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Another video of root cells of the Arabidopsis S line transformed with pSPX1::MS2×128 and MCP–GFP, after receiving a Pi-rich solution at time t = 0 min. MIP from a time-lapse video recorded in 3D (200 z planes). Acquisitions lasted 54 min.

Supplementary Video 6

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Magnification deriving from Supplementary Video 5.

Supplementary Video 7

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Magnification deriving from Supplementary Video 5.

Supplementary Video 8

Transcriptional repression of the pSPX1::MS2×128 reporter triggered by Pi resupply. Video of root cells of the Arabidopsis J line transformed with pSPX1::MS2×128 and MCP–GFP, after receiving a Pi-rich solution at time t = 0 min. MIP from a time-lapse video recorded in 3D (200 z planes). Acquisitions lasted 54 min.

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Hani, S., Cuyas, L., David, P. et al. Live single-cell transcriptional dynamics via RNA labelling during the phosphate response in plants. Nat. Plants 7, 1050–1064 (2021). https://doi.org/10.1038/s41477-021-00981-3

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