Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

CLASP balances two competing cell division plane cues during leaf development

Abstract

Starting as small, densely packed boxes, leaf mesophyll cells expand to form an intricate mesh of interconnected cells and air spaces, the organization of which dictates the internal surface area of the leaf for light capture and gas exchange during photosynthesis. Despite their importance, little is known about the basic patterns of mesophyll cell division, and how they contribute to cell and intercellular space organization. To address this, we tracked divisions within individual cell lineages in three dimensions over time in Arabidopsis spongy mesophyll. We found that early on, successive cell division planes switch their orientation such that each new cell wall intersects the previous at a right angle, creating a new multi-cell junction (the intersection of three or more cells). These junctions then open to create intercellular spaces. During subsequent enlargement of the spaces, the division planes of the surrounding cells show an increasing tendency to tilt in the direction of their adjacent intercellular spaces. This disrupts the alternating pattern, and by extension, halts the initiation of new multi-cell junctions and intercellular spaces, but allows the expansion of existing spaces. Both division patterns are specified before mitosis by the orientation of interphase cortical microtubules, which gradually narrow to form a preprophase band in the same orientation to establish the future plane of cell division. In the absence of the microtubule-associated protein CLASP, the early alternating division plane and microtubule patterns are compromised, whereas space-oriented divisions are exacerbated. This results in large distortions of the topological relations between cells and intercellular spaces, as well as changes in their relative abundance. Our data reveal the existence of two competing cell division mechanisms that are balanced by CLASP to specify the distribution of cells and intercellular spaces in spongy mesophyll tissue.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Alternating cell divisions drive intercellular space formation in wild-type spongy mesophyll.
Fig. 2: Intercellular space-oriented cell divisions.
Fig. 3: Organization of interphase MTs predicts the subsequent cell divisions.
Fig. 4: CLASP localizes to cell edges and is required for the establishment of perpendicular PCAs.
Fig. 5: CLASP is required for the alternating division pattern.
Fig. 6: Cell division patterns in wild-type and clasp-1 spongy mesophyll cells.
Fig. 7: Influence of clasp-1 cell division defects on cell and intercellular space topologies in spongy mesophyll.
Fig. 8: Cell division plane defects in clasp-1 lead to a less organized topology of cell–cell contacts.

Data availability

The plasmids, Arabidopsis lines and confocal stacks used in this study are available from the corresponding author upon request. Source data are provided with this paper.

References

  1. Hashimoto, T. Microtubules in plants. Arabidopsis Book 13, e0179 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Lloyd, C. & Chan, J. Microtubules and the shape of plants to come. Nat. Rev. Mol. Cell Biol. 5, 13–23 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Wasteneys, G. O. Microtubule organization in the green kingdom: chaos or self-order? J. Cell Sci. 115, 1345–1354 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Ehrhardt, D. W. & Shaw, S. L. Microtubule dynamics and organization in the plant cortical array. Annu. Rev. Plant Biol. 57, 859–875 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Wasteneys, G. O. & Ambrose, J. C. Spatial organization of plant cortical microtubules: close encounters of the 2D kind. Trends Cell Biol. 19, 62–71 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Baskin, T. I. Anisotropic expansion of the plant cell wall. Annu. Rev. Cell Dev. Biol. 21, 203–222 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Paradez, A., Wright, A. & Ehrhardt, D. W. Microtubule cortical array organization and plant cell morphogenesis. Curr. Opin. Plant Biol. 9, 571–578 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Peaucelle, A., Wightman, R. & Höfte, H. The control of growth symmetry breaking in the Arabidopsis hypocotyl. Curr. Biol. 25, 1746–1752 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Mineyuki, Y. in International Review of Cytology Vol. 187 (ed. Jeon, K. W.) 1–49 (Elsevier, 1999).

  10. Ambrose, J. C. & Cyr, R. Mitotic spindle organization by the preprophase band. Mol. Plant 1, 950–960 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Chan, J., Calder, G., Fox, S. & Lloyd, C. Localization of the microtubule end binding protein EB1 reveals alternative pathways of spindle development in Arabidopsis suspension cells. Plant Cell 17, 1737–1748 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Livanos, P. & Müller, S. Division plane establishment and cytokinesis. Annu. Rev. Plant Biol. 70, 239–267 (2019).

    Article  PubMed  Google Scholar 

  13. Müller, S. Universal rules for division plane selection in plants. Protoplasma 249, 239–253 (2012).

    Article  PubMed  Google Scholar 

  14. Rasmussen, C. G., Wright, A. J. & Müller, S. The role of the cytoskeleton and associated proteins in determination of the plant cell division plane. Plant J. 75, 258–269 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Müller, S., Wright, A. J. & Smith, L. G. Division plane control in plants: new players in the band. Trends Cell Biol. 19, 180–188 (2009).

    Article  PubMed  CAS  Google Scholar 

  16. Besson, S. & Dumais, J. Universal rule for the symmetric division of plant cells. Proc. Natl Acad. Sci. USA 108, 6294–6299 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Carter, R., Sanchez-Corrales, Y. E., Hartley, M., Grieneisen, V. A. & Maree, A. F. M. Pavement cells and the topology puzzle. Development 144, 4386–4397 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Louveaux, M., Julien, J.-D., Mirabet, V., Boudaoud, A. & Hamant, O. Cell division plane orientation based on tensile stress in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 113, E4294–E4303 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Reddy, G. V. Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana. Development 131, 4225–4237 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Shapiro, B. E., Tobin, C., Mjolsness, E. & Meyerowitz, E. M. Analysis of cell division patterns in the Arabidopsis shoot apical meristem. Proc. Natl Acad. Sci. USA 112, 4815–4820 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yoshida, S. et al. Genetic control of plant development by overriding a geometric division rule. Dev. Cell 29, 75–87 (2014).

    Article  CAS  PubMed  Google Scholar 

  22. MacAdam, J. W., Volenec, J. J. & Nelson, C. J. Effects of nitrogen on mesophyll cell division and epidermal cell elongation in tall fescue leaf blades. Plant Physiol. 89, 549–556 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Pyke, K. A., Marrison, J. L. & Leech, A. M. Temporal and spatial development of the cells of the expanding first leaf of Arabidopsis thaliana (L.) Heynh. J. Exp. Bot. 42, 1407–1416 (1991).

    Article  Google Scholar 

  24. Dorca-Fornell, C. et al. Increased leaf mesophyll porosity following transient retinoblastoma-related protein silencing is revealed by microcomputed tomography imaging and leads to a system-level physiological response to the altered cell division pattern. Plant J. 76, 914–929 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Jeffree, C. E., Dale, J. E. & Fry, S. C. The genesis of intercellular spaces in developing leaves of Phaseolus vulgaris L. Protoplasma 132, 90–98 (1986).

    Article  Google Scholar 

  26. Kolloffel, C. & Linssen, P. W. T. The formation of intercellular spaces in the cotyledons of developing and germinating pea seeds. Protoplasma 120, 12–19 (1984).

    Article  Google Scholar 

  27. Zhang, L., McEvoy, D., Le, Y. & Ambrose, C. Live imaging of microtubule organization, cell expansion, and intercellular space formation in Arabidopsis leaf spongy mesophyll cells. Plant Cell 33, 623–641 (2021).

    Article  PubMed  Google Scholar 

  28. Gunning, B. E. S. & Sammut, M. Rearrangements of microtubules involved in establishing cell-division planes start immediately after DNA-synthesis and are completed just before mitosis. Plant Cell 2, 1273–1282 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hamant, O. et al. Developmental patterning by mechanical signals in Arabidopsis. Science 322, 1650–1655 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Kimata, Y. et al. Cytoskeleton dynamics control the first asymmetric cell division in Arabidopsis zygote. Proc. Natl Acad. Sci. USA 113, 14157–14162 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rasmussen, C. G. & Bellinger, M. An overview of plant division-plane orientation. New Phytol. 219, 505–512 (2018).

    Article  PubMed  Google Scholar 

  32. Ambrose, C., Allard, J. F., Cytrynbaum, E. N. & Wasteneys, G. O. A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis. Nat. Commun. 2, 430 (2011).

    Article  PubMed  CAS  Google Scholar 

  33. Chakrabortty, B. et al. A plausible microtubule-based mechanism for cell division orientation in plant embryogenesis. Curr. Biol. 28, 3031–3043.e2 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Ruan, Y. et al. The microtubule-associated protein CLASP sustains cell proliferation through a brassinosteroid signaling negative feedback loop. Curr. Biol. 28, 2718–2729.e5 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. Bichet, A., Desnos, T., Turner, S., Grandjean, O. & Höfte, H. BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis. Plant J. 25, 137–148 (2001).

    Article  CAS  PubMed  Google Scholar 

  36. Kawamura, E. et al. MICROTUBULE ORGANIZATION 1 regulates structure and function of microtubule arrays during mitosis and cytokinesis in the Arabidopsis root. Plant Physiol. 140, 102–114 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Burk, D. H., Liu, B., Zhong, R., Morrison, W. H. & Ye, Z. H. A katanin-like protein regulates normal cell wall biosynthesis and cell elongation. Plant Cell 13, 807–827 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Gallagher, K. & Smith, L. G. Roles for polarity and nuclear determinants in specifying daughter cell fates after an asymmetric cell division in the maize leaf. Curr. Biol. 10, 1229–1232 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Buschmann, H. et al. Microtubule-associated AIR9 recognizes the cortical division site at preprophase and cell-plate insertion. Curr. Biol. 16, 1938–1943 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Walker, K. L., Müller, S., Moss, D., Ehrhardt, D. W. & Smith, L. G. Arabidopsis TANGLED identifies the division plane throughout mitosis and cytokinesis. Curr. Biol. 17, 1827–1836 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Azimzadeh, J. et al. Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin. Plant Cell 20, 2146–2159 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Traas, J. et al. Normal differentiation patterns in plants lacking microtubular preprophase bands. Nature 375, 676–677 (1995).

    Article  CAS  Google Scholar 

  43. Gilliland, L. U., Pawloski, L. C., Kandasamy, M. K. & Meagher, R. B. Arabidopsis actin gene ACT7 plays an essential role in germination and root growth. Plant J. 33, 319–328 (2003).

    Article  CAS  PubMed  Google Scholar 

  44. Louveaux, M. & Hamant, O. The mechanics behind cell division. Curr. Opin. Plant Biol. 16, 774–779 (2013).

    Article  PubMed  Google Scholar 

  45. Belteton, S. A. et al. Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern. Nat. Plants 7, 826–841 (2021).

    Article  CAS  PubMed  Google Scholar 

  46. Bidhendi, A. J., Altartouri, B., Gosselin, F. P. & Geitmann, A. Mechanical stress initiates and sustains the morphogenesis of wavy leaf epidermal cells. Cell Rep. 28, 1237–1250.e6 (2019).

    Article  CAS  PubMed  Google Scholar 

  47. Sampathkumar, A. et al. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells. eLife 3, e01967 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  48. Panteris, E. & Galatis, B. The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments: research review. New Phytol. 167, 721–732 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Ambrose, C. et al. CLASP interacts with sorting nexin 1 to link microtubules and auxin transport via PIN2 recycling in Arabidopsis thaliana. Dev. Cell 24, 649–659 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Halat, L., Gyte, K. & Wasteneys, G. The microtubule-associated protein CLASP is translationally regulated in light-dependent root apical meristem growth. Plant Physiol. 184, 2154–2167 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Al-Bassam, J. & Chang, F. Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol. 21, 604–614 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Goodson, H. V. & Jonasson, E. M. Microtubules and microtubule-associated proteins. Cold Spring Harb. Perspect. Biol. 10, a022608 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Hamada, T. Microtubule organization and microtubule-associated proteins in plant cells. Int. Rev. Cell Mol. Biol. 312, 1–52 (2014).

    Article  PubMed  Google Scholar 

  54. Maiato, H., Khodjakov, A. & Rieder, C. L. Drosophila CLASP is required for the incorporation of microtubule subunits into fluxing kinetochore fibres. Nat. Cell Biol. 7, 42–47 (2005).

    Article  CAS  PubMed  Google Scholar 

  55. Bratman, S. V. & Chang, F. Stabilization of overlapping microtubules by fission yeast CLASP. Dev. Cell 13, 812–827 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ambrose, J. C., Shoji, T., Kotzer, A. M., Pighin, J. A. & Wasteneys, G. O. The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division. Plant Cell19, 2763–2775 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Kirik, V. et al. CLASP localizes in two discrete patterns on cortical microtubules and is required for cell morphogenesis and cell division in Arabidopsis. J. Cell Sci. 120, 4416–4425 (2007).

    Article  CAS  PubMed  Google Scholar 

  58. Schaefer, E. et al. The preprophase band of microtubules controls the robustness of division orientation in plants. Science 356, 186–189 (2017).

    Article  CAS  PubMed  Google Scholar 

  59. Eng, R. C. et al. KATANIN and CLASP function at different spatial scales to mediate microtubule response to mechanical stress in Arabidopsis cotyledons. Curr. Biol. 31, 3262–3274 (2021).

    Article  CAS  PubMed  Google Scholar 

  60. Ambrose, J. C. & Wasteneys, G. O. CLASP modulates microtubule–cortex interaction during self-organization of acentrosomal microtubules. Mol. Biol. Cell 19, 4730–4737 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Le, P. Y. & Ambrose, C. CLASP promotes stable tethering of endoplasmic microtubules to the cell cortex to maintain cytoplasmic stability in Arabidopsis meristematic cells. PLoS ONE 13, e0198521 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Hamant, O., Inoue, D., Bouchez, D., Dumais, J. & Mjolsness, E. Are microtubules tension sensors? Nat. Commun. 10, 2360 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Evert, R. F. Esau’s Plant Anatomy: Meristems, Cells, and Tissues of the Plant Body: Their Structure, Function, and Development (Wiley, 2006); https://doi.org/10.1002/0470047380

  64. Schüepp, O. Untersuchungen über Wachstum und Formwechsel von Vegetationspunkten. Jb.Bot. 57 (1916).

  65. Gunning, B. E. S., Hughes, J. E. & Hardham, A. R. Formative and proliferative cell divisions, cell differentiation, and developmental changes in the meristem of Azolla roots. Planta 143, 121–144 (1978).

    Article  CAS  PubMed  Google Scholar 

  66. Rudall, P. J. & Knowles, E. V. W. Ultrastructure of stomatal development in early-divergent angiosperms reveals contrasting patterning and pre-patterning. Ann. Bot. 112, 1031–1043 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Bergmann, D. C. & Sack, F. D. Stomatal development. Annu. Rev. Plant Biol. 58, 163–181 (2007).

    Article  CAS  PubMed  Google Scholar 

  68. Serna, L., Torres-Contreras, J. & Fenoll, C. Clonal analysis of stomatal development and patterning in Arabidopsis leaves. Dev. Biol. 241, 24–33 (2002).

    Article  CAS  PubMed  Google Scholar 

  69. Minc, N. & Piel, M. Predicting division plane position and orientation. Trends Cell Biol. 22, 193–200 (2012).

    Article  PubMed  Google Scholar 

  70. Le, J., Mallery, E. L., Zhang, C., Brankle, S. & Szymanski, D. B. Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2. Curr. Biol. 16, 895–901 (2006).

    Article  CAS  PubMed  Google Scholar 

  71. Whittington, A. T. et al. MOR1 is essential for organizing cortical microtubules in plants. Nature 411, 610–613 (2001).

    Article  CAS  PubMed  Google Scholar 

  72. Boudaoud, A. et al. FibrilTool, an imagej plug-in to quantify fibrillar structures in raw microscopy images. Nat. Protoc. 9, 457–463 (2014).

    Article  CAS  PubMed  Google Scholar 

  73. Fonck, E. et al. Effect of aging on elastin functionality in human cerebral arteries. Stroke 40, 2552–2556 (2009).

    Article  CAS  PubMed  Google Scholar 

  74. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  75. Waskom, M. seaborn: statistical data visualization. J. Open Source Softw. 6, 3021 (2021).

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank D. McEvoy (University of Saskatchewan) for a critical reading of the manuscript. This work was supported by the Natural Sciences and Engineering Research Council (NSERC) Discovery Grant 298264-2009, the University of Saskatchewan new faculty start-up funds and the University of Saskatchewan Biology Department graduate student scholarships.

Author information

Authors and Affiliations

Authors

Contributions

L.Z. and C.A. conceptualized the project. L.Z. developed the methodologies and performed the experiments. L.Z. and C.A. analysed the data and wrote the manuscript. C.A. acquired the funding.

Corresponding author

Correspondence to Chris Ambrose.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Plants thanks Olivier Hamant, Markus Grebe and Bo Liu for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Source data

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

Source Data Fig. 7

Statistical source data.

Source Data Fig. 8

Statistical source data.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, L., Ambrose, C. CLASP balances two competing cell division plane cues during leaf development. Nat. Plants 8, 682–693 (2022). https://doi.org/10.1038/s41477-022-01163-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41477-022-01163-5

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing