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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

Abstract

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

Introduction

Plant cell walls undergo dynamic changes during cell expansion and development1,2,3. Maintaining cell wall integrity is critical for plant cell adhesion during growth and development, as well as for the response to environmental signals. Although visualizing cell wall dynamics of living cells over a long period of time is critical to understanding how cell adhesion is maintained during development and adaptation to environmental changes, current methods for directly observing cell wall dynamics are still challenging.

Time-lapse imaging of cellular ....

Protocol

NOTE: See the Table of Materials for the list of materials and equipment and Table 1 for the list of solutions to be used in this protocol.

1. Preparation of plants for glass bottom dishes

  1. Grow the moss protonemal tissue in BCDAT agar medium in a 13 cm Petri dish under constant white light (~50 µmol m-2s-1) for 7 days at 25 °C in the growth chamber.
  2. Filter-sterilize the calcofluor white and.......

Representative Results

This method allows the observation of cell wall dynamics during development in wild type and ggb mutants (Figure 1). The results showed that regions with less thickening of the cell wall correlate with the cell expansion/branching sites, allowing for the prediction of expansion/branching sites in the wild type (Figure 1A). The surface of the cell walls in ggb mutants was torn apart during development due to uncontrolled cell expansion

Discussion

Time-lapse 3-D reconstruction, or 4-D imaging, is a powerful tool for observing the dynamics of cellular morphology during developmental processes. In this protocol, by mixing the calcofluor white in the medium, the dynamics of 3-D cellular morphology can be observed in the moss P. patens. Using this method, we observed that the surface of cell walls in ggb mutants are torn apart during development3. Moreover, the reduced thickening of cell walls is correlated with the cell expan.......

Acknowledgements

The authors thank Dr. Soucy Patricia and Betty Nunn at the University of Louisville for assistance with the confocal microscope. This work was funded by the National Science Foundation (1456884 to M.P.R.) and by a National Science Foundation Cooperative Agreement (1849213 to M.P.R.).

....

Materials

NameCompanyCatalog NumberComments
3-mm-thick red plastic light filter Mitsubishi no.102
27 mm diameter glass base dish Iwaki3930-035
Agar SigmaA6924
Calcofluor white  Sigma18909-100ML-FCalcofluor White M2R, 1 g/L and Evans blue, 0.5 g/L
Confocal microscope Nikon A1

NIS element software; .nd2 file in NIS-elements Viewer, download from https://www.microscope.healthcare.nikon.
com/en_AOM/products/software/nis-elements/viewer

Fluorescence microscope Nikon TE200 Equipped with a DS-U3 camera;
Gellan gum Nacali Tesque12389-96
Plant Growth ChambersSANYO Sanyo MLR-350H
Sterilized syringe 0.22 μm filterMilliporeSLGV033RS

References

  1. Anderson, C. T., Kieber, J. J. Dynamic construction, perception, and remodeling of plant cell walls. Annual Review of Plant Biology. 71, 39-69 (2020).
  2. Vaahtera, L., Schulz, J., Hamann, T. Ce....

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