Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 1

Fig. 1 | Monitoring of cortical microtubules (CMTs) in root epidermal cells.

A. Root tip of Arabidopsis with distinct growth zones marked: meristematic (MZ, green), transition (TZ, blue), elongation (EZ, pink) and differentiation (DZ, yellow). Double arrow indicates the first expanded cortex cell considered as a start point of the TZ, which encompasses epidermal cells smaller than 45 µm. Epidermal cells reaching a length of more than 45 µm prior termination of the elongation are in the EZ. Scale bar 25 µm.

B. CMTs visualized by the MAP4-GFP reporter (left) and scheme of the CMT orientations (right) in epidermal cells of distinct growth zones (TZ, EZ and DZ). Individual cells monitored at two time points (0 and 60 min). Dark blue arrows mark the cell expansion direction. Scale bar 10 µm.

C. Histograms of the CMT orientation distributions. Orientation was measured as an angle between CMTs and the longitudinal root growth axis, with 0º, 45º and 90º corresponding to longitudinal, oblique and transversal orientations, respectively. The proportion of CMTs in a certain orientation is calculated per cell (n = 15-20 cells per root growth zone with 6-9 roots in 4 independent replicates).

D. Analysis of the CMT plus-end growth with the EB1b-GFP reporter. CMT plus-end growth rates (μm/min) measured by tracking the EB1b-GFP marker for 20-30 min in epidermal root cells of three growth zones. In the boxplots, the center lines show medians; box limits indicate the 25th and 75th percentiles as determined by the Graphpad software; whiskers span minimum to maximum values, individual data points are represented by dots. ** P < 0.01, **** P < 0.0001by the Student's t-test. The number of CMT plus-end events (EB1b-GFP) tracked per cell was 20-100/min, of which the growth rate average is represented as a single dot; 3-5 cells per root growth zone, 3-5 roots analyzed per biological replicate in 3 independent experiments. On the right, single trajectories of EB1b-GFP signal tracked over 60 sec in root epidermal cells at different growth zones. Scale bar 0.5 µm.

E. Z-stack maximum image projection of the EB1b-GFP plus-end reporter tracked over 30 sec, in epidermal cells at different growth zones. Scale bars 10 µm.

F. CMTs visualized by MAP4-GFP in root epidermal cells at the TZ, the EZ and the DZ prior (0) and 60 min after treatment with 1 µm oryzalin. Scale bar 10 µm.

G. Quantification of the MAP4-GFP CMTs reporter signal in wild-type root epidermal cells at the TZ, the EZ and the DZ zone treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and cytokinin (CK; 10 µM 6-benzylaminopurine, BAP) and oryzalin (1 µM) (dark grey box). For double treatments, roots pretreated for 60 min with CK prior to transfer to medium supplemented with both compounds. Boxplots represent ratio between mean fluorescence intensity (arbitrary units) measured in epidermal cells at 60 and 0 min.
The center lines show the medians and the box limits indicate the 25th and 75th percentiles; whiskers span the minimal to maximal values, individual data points are represented by dots. Ratio close to 1 (segmented line) corresponds to the unchanged MAP-GFP signal for 60 min. (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates).