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Live-cell imaging of the cytoskeleton in elongating cotton fibres

Abstract

Cotton (Gossypium hirsutum) fibres consist of single cells that grow in a highly polarized manner, assumed to be controlled by the cytoskeleton1,2,3. However, how the cytoskeletal organization and dynamics underpin fibre development remains unexplored. Moreover, it is unclear whether cotton fibres expand via tip growth or diffuse growth2,3,4. We generated stable transgenic cotton plants expressing fluorescent markers of the actin and microtubule cytoskeleton. Live-cell imaging revealed that elongating cotton fibres assemble a cortical filamentous actin network that extends along the cell axis to finally form actin strands with closed loops in the tapered fibre tip. Analyses of F-actin network properties indicate that cotton fibres have a unique actin organization that blends features of both diffuse and tip growth modes. Interestingly, typical actin organization and endosomal vesicle aggregation found in tip-growing cell apices were not observed in fibre tips. Instead, endomembrane compartments were evenly distributed along the elongating fibre cells and moved bi-directionally along the fibre shank to the fibre tip. Moreover, plus-end tracked microtubules transversely encircled elongating fibre shanks, reminiscent of diffusely growing cells. Collectively, our findings indicate that cotton fibres elongate via a unique tip-biased diffuse growth mode.

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Fig. 1: F-actin architectures and dynamics in cotton fibre cells at different developmental stages.
Fig. 2: Automated network extraction analysis shows that the F-actin organization in cotton fibres blends features of that in tip- and diffuse-growth cells.
Fig. 3: Microtubule array organization, tracked by the plus-end tracking protein EB1b, in cotton fibre cells during development.
Fig. 4: Apical F-actin, cytoplasmic trafficking features and a proposed tip-biased diffuse mode for fibre elongation.

Data availability

All data are available from the corresponding authors upon request.

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Acknowledgements

We thank G.-X. Xia (Institute of Microbiology, Chinese Academy of Sciences) for providing valuable assistance and insightful suggestions about the project. We also thank D. Delmer (University of California, Davis) for critical comments and valuable suggestions on the manuscript. We are grateful to L. Su (Institute of Microbiology, Chinese Academy of Sciences) for providing technical assistance in microscopy We also appreciate the technical support of the UltraView Vox system from L. Jiao in Perkin Elmer. This study was supported by the National Key Research and Development Program of China 2016YFD0100505 and 2016YFD0100306, by the National Science Foundation of China under grant no. 31371676, by the startup fund of ‘One Hundred Talents’ programme of the Chinese Academy of Sciences, by the National Natural Science Foundation of China (31601350), by the National Major Project for Developing New GM Crops (2016ZX08005) and by the grants from the State Key Laboratory of Plant Genomics. S.P. was funded by a R@MAP Professorship at University of Melbourne, by a Future Fellowship grant (FT160100218) and by an IRRTF-RNC grant via University of Melbourne.

Author information

Authors and Affiliations

Authors

Contributions

Y.Y. performed live-cell imaging on cytoskeleton dynamics, prepared figures and videos, and generated stable transgenic cotton plants expressing the mCherry–EB1b reporter. S.W. conducted stable cotton transformation for the ABD2–GFP reporter, and field experiments for the screening and propagation of the cotton marker lines. J.N. analysed F-actin network properties of cotton fibres, hypocotyls and root hairs. G.W. analysed microtubule and F-actin dynamics, prepared figures and videos, developed the code for generating the progressively increasing mCherry–EB1b series and designed the working model. Z.F. made the mCherry–EB1b expression vector. A.M., L.H., Y.M., H.W., X.Z., J.T. and L.D. provided essential technical assistance. Z.N. provided guidance in the analysis of network properties. S.P. supervised the analysis of network properties. Z.K. conceived the project, interpreted the data and wrote the manuscript with the input of other authors. Z.K., S.P., Z.N. and J.N. revised the article.

Corresponding authors

Correspondence to Staffan Persson or Zhaosheng Kong.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figuress 1–6, legends for Supplementary Videos, and Tables 1 and 2.

Reporting Summary

Supplementary Video 1

The 3D reconstruction of F-actin organization in cotton fibre cells at 0 DPA versus 2 DPA. Related to Supplementary Fig. 2a,b. Representative videos are shown based on data from six plants in three independent experiments, respectively.

Supplementary Video 2

The 3D reconstruction of F-actin organization in cotton fibre cells at 0 DPA, 2 DPA and 12 DPA, respectively. Related to Supplementary Fig. 2c–h. Representative videos for fibres at 0 DPA and 2 DPA are shown based on data from six plants in three independent experiments, respectively. The representative video for fibres at 12 DPA is shown based on data from five plants in three independent experiments.

Supplementary Video 3

The F-actin dynamics in cotton fibre cells at 0 DPA. Related to Fig. 1a,d. The yellow box indicates a severing event at 0 DPA. Scale bar, 10 μm. The representative video is shown based on data from six plants in three independent experiments.

Supplementary Video 4

The F-actin dynamics in cotton fibre cells at 2 DPA. Related to Fig. 1b,e,f. The cyan box indicates a bundling event and the magenta box illustrating a de-polymerization event at 2 DPA. Scale bar, 10 μm. The representative video is shown based on data from six plants in three independent experiments.

Supplementary Video 5

The 3D reconstruction of EB1b illustrating the plus-end of growing microtubules in elongating cotton fibres. Related to Supplementary Fig. 4b,c. The representative video is shown based on data from five plants in three independent experiments.

Supplementary Video 6

The EB1b movement and trajectory projection illustrating the distribution of cortical microtubules in cotton leaf guard cells. Related to Supplementary Fig. 4d,e. Scale bars, 10 μm. Representative videos are shown based on data from six plants in three independent experiments.

Supplementary Video 7

The EB1b movement and trajectory projection illustrating the cortical microtubule networks in cotton fibre cells at 0 DPA. Related to Fig. 3a,c. Scale bars, 10 μm. Representative videos are shown based on data from eight plants in three independent experiments.

Supplementary Video 8

The EB1b movement and trajectory projection illustrating the cortical microtubule networks in cotton fibre cells at 2 DPA. Related to Fig. 3b,d. Scale bars, 10 μm. Representative videos are shown based on data from eight plants in three independent experiments.

Supplementary Video 9

3D reconstruction illustrating transverse microtubule arrays encircling elongating cotton fibres at 2 DPA, with a microtubule-depleted zone at the fibre apex. Related to Supplementary Fig. 6. The representative video is shown based on data from five plants in three independent experiments.

Supplementary Video 10

F-actin architecture and dynamics in the tip areas of elongating cotton fibre cells. F-actin strings extend along the long axis of fibre cells to form closed loops (indicated by yellow arrows) in the fibre tip. Related to Fig. 4a. Scale bars, 10 μm. The representative video is shown based on data from five plants in three independent experiments.

Supplementary Video 11

Secretory vesicles are evenly distributed in elongating fibre cells. Related to Fig. 4b. Scale bar, 10 μm. Representative videos are shown based on data from five plants in three independent experiments.

Supplementary Video 12

Vesicles undergo bi-directional movements from the fibre shank to the tip. Related to Fig. 4c. The arrows track the movements of vesicles following cytoplasmic streaming. The cyan circle highlights one single vesicle moving away from the tip and the yellow circle highlights one single vesicle moving towards the tip. Scale bar, 10 μm. The representative video is shown based on data from five plants in three independent experiments.

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Yu, Y., Wu, S., Nowak, J. et al. Live-cell imaging of the cytoskeleton in elongating cotton fibres. Nat. Plants 5, 498–504 (2019). https://doi.org/10.1038/s41477-019-0418-8

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