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Visualization of cellulose synthases in Arabidopsis secondary cell walls

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Science  09 Oct 2015:
Vol. 350, Issue 6257, pp. 198-203
DOI: 10.1126/science.aac7446
  • Fig. 1 CESA7 and microtubules are constrained to secondary cell wall domains during protoxylem development.

    (A) Early development: YFP::CESA7-labeled CSCs and RFP::TUB6-labeled microtubules localize diffusely across the plasma membrane. (B) Mid-development: Narrow microtubule bundles lie underneath tight tracks of membrane-localized CSCs. (C) Late development: Apparent concentrations of YFP::CESA7 and RFP::TUB6 at the edges of forming secondary cell walls are revealed as uniform signal in subcortical optical sections. YFP::CESA7-labeled Golgi (arrowhead) are visible because of the increased depth of imaging through secondary cell wall thickenings. (D) TEM micrographs highlight plasma membrane (PM) curvature over secondary cell walls (2CW), lined by cortical microtubule (MT) bundles. Scale bars, 10 μm [(A) to (C)], 500 nm (D).

  • Fig. 2 Secondary cell wall CSCs have a higher velocity than primary cell wall CSCs during peak cellulose deposition.

    (A and B) CSC tracks in single frames and time projections for primary cell wall (A) and secondary cell wall (B) visualized using GFP::CESA3 and YFP::CESA7, respectively. Kymographs sampled along the yellow lines show CSC trajectories over time, from which CSC velocity was calculated. Scale bars, 10 μm. (C and D) Histograms of GFP::CESA3 (C) and YFP::CESA7 (D) velocities calculated from kymograph analysis. (E) Box plot of CSC velocities across stages of xylem cell development. Means with different letters represent statistically significant differences (Tukey’s pairwise comparison, P < 0.01). For each developmental stage, 480 CSC velocities were measured from 12 cells in four plants. In (D), 1440 velocities were pooled from all developmental stages. In (E), velocities were averaged for each cell before analysis.

  • Fig. 3 Golgi and SmaCCs densely populate and rapidly deliver CSCs to domains of secondary cell wall formation.

    (A) Fluorescence recovery after photobleaching (FRAP) of YFP::CESA7 in the boxed area, overlying a secondary cell wall thickening. Abundant Golgi-independent SmaCCs (red and blue arrows) rapidly repopulate bleached regions. Additionally, Golgi (arrowhead) and closely associated SmaCCs (yellow arrow) can be seen moving from one secondary cell wall band and pausing at another. (B) FRAP and kymograph analyses demonstrating insertion at the plasma membrane of at least two YFP::CESA7-labeled CSCs from a SmaCC (yellow arrow). After the Golgi (arrowhead) moves away, the signal from the SmaCC splits into two distinct punctae with steady velocities (red and orange arrows). (C) TEM micrographs of cytoplasm around secondary cell walls showing a diversity of closely associated vesicles. Trans-Golgi networks (arrows), secretory vesicle clusters (arrowheads), and electron-lucent vesicles (asterisks) are indicated. Scale bars, 5 μm (A), 2.5 μm (B), 500 nm (C).

  • Fig. 4 Secondary cell wall CSC distribution at the plasma membrane is disorganized by the loss of microtubules after oryzalin treatment while CSC delivery and motility are unaffected.

    (A) VND7::GR-induced cells expressing YFP::CESA7 and RFP::TUB6 after oryzalin treatment. Plasma membrane–localized secondary cell wall CSCs follow aberrant tracks in the absence of microtubules. (B) CSC velocities were not significantly different between oryzalin- and DMSO-treated cells (Student’s t test, P = 0.49). For each condition, 40 CSC velocities were averaged for each of 12 cells in four plants. (C) FRAP of YFP::CESA7 signal revealed insertion of CSCs at the plasma membrane after microtubule loss (arrows). Scale bars, 10 μm [(A) and (B)].

Supplementary Materials

  • Visualization of cellulose synthases in Arabidopsis secondary cell walls

    Y. Watanabe, M. J. Meents, L. M. McDonnell, S. Barkwill, A. Sampathkumar, H. N. Cartwright, T. Demura, D. W. Ehrhardt, A.L. Samuels, S. D. Mansfield

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S5
    • Tables S1 and S2
    • Captions for movies S1 to S6

    Images, Video, and Other Other Media

    Movie S1
    Distribution and motility of YFP::CESA7 in the cell membrane of VND7::GR induced seedlings at mid-development of secondary cell wall formation.
    Time lapse of VND7::GR induced cells with YFP::CESA7 and RFP::TUB6. Linear, bi-directional steady movement of YFP::CESA7 particles (arrowheads) are observed in tight domains of secondary cell wall formation. These domains are closely associated with underlying cortical RFP::TUB6 labeled microtubules. Movie acquired over 5 min at 5 sec intervals, and processed as described in the materials and methods. Scale bar: 10 μm.
    Movie S2
    Golgi and SmaCCs containing YFP::CESA7 rapidly move between and pause at domains of secondary cell wall formation.
    Time lapse of VND7::GR induced cells with YFP::CESA7. Numerous intracellular compartments containing YFP::CESA7 are observed including Golgi (red arrows), Golgi-independent SmaCCs (blue arrows) and Golgi-associated SmaCCs (yellow arrows). Compartments are observed to move quickly between domains and pause at domains of secondary cell wall formation. Movie acquired over 5 min at 5 sec intervals. Scale bar: 10 μm.
    Movie S3
    Coordination between YFP::CESA7 containing Golgi and SmaCCs can be transient.
    FRAP of a secondary cell wall thickening reveals a YFP::CESA7 containing Golgi body (yellow arrowhead) and closely associated SmaCC (red arrowhead) moving rapidly from the lower secondary cell wall band and approach and pause at the upper secondary cell wall domain. The Golgi body eventually moves on, while the SmaCC remains stationary, presumably representing an insertion event into the plasma membrane. Movie acquired over 7.5 min at 5 sec intervals. Scale bar: 2.5 μm.
    Movie S4
    Delivery of at least two YFP::CESA7 containing complexes by a SmaCC.
    FRAP of a plasma membrane surrounding secondary cell wall thickening reveals YFP::CESA7 containing Golgi and closely associated SmaCC pause. The Golgi body eventually moves on while the SmaCC remains stationary and splits into two distinct particles with linear trajectories and steady velocities (arrowheads) presumably representing an insertion event of at least two CSCs to the plasma membrane. Movie acquired over 7.5 min at 5 sec intervals. Scale bar: 2.5 μm.
    Movie S5
    Loss of secondary cell wall CSC organization at the plasma membrane due to near complete depolymerization of microtubules with oryzalin treatment.
    Cells were treated with 20 μM oryzalin for 6 hrs prior to induction with dexamethasone. Loss of cortical microtubules prior to and during induction results in YFP::CESA7 labeled complexes at the plasma membrane lose their tight annular banding pattern as observed in the DMSO control. Instead disorganized „swarms‟ of particles are observed at the plasma membrane. Movie acquired over 5 min., images acquired every 5 sec intervals. Scale Bar: 10 μm.
    Movie S6
    Insertion events of secondary cell wall CSC complexes to the plasma membrane are unaffected by depolymerization of microtubules s by oryzalin treatment.
    Time lapse movie of a FRAP experiment of oryzalin treated induced VND7::GR cells reveals that insertion of YFP::CESA7 labeled complexes at the plasma membrane (arrowheads) still occur without the presence of microtubules. Movie acquired over 7.5 min, images acquired every 5 sec Scale Bar: 5 μm

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