Phytohormone cytokinin guides microtubule dynamics during cell progression from proliferative to differentiated stage (2020)
Figure 3

Fig. 3 | The AHK4/CRE1 receptor mediates the cytokinin effects on CMTs.

A-D. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant at the transition (TZ) and the elongation (EZ) zones at time point 0 and 60 min after mock (DMSO) (A), cytokinin (CK) (B), auxin (C) and CK plus auxin (D) treatments. As CK and auxin sources 10 µM BAP and 0.1 µM NAA were used, respectively. For the double CK plus auxin treatment roots were pretreated for 60 min with CK, whereafter they were transferred to medium supplemented with both compounds. Scale bar 10 µm.

E. Histograms of CMT orientation distributions at time points 0 and 60 min in epidermal cells of cre1-12 at the TZ and the EZ treated as for (A-D). n = 14-29 cells per root growth zone in 4-8 roots per condition in 3 independent replicates.

F, G. Analysis of the CMT plus-end growth with the EB1b-GFP reporter in cre1-12. F. Z-stack maximum image projection of EB1b-GFP tracked for 30 seconds (on the left) and single trajectories of EB1b-GFP signal tracked over 60 sec (on the right) in root epidermal cells of the EZ in the wild type and cre1-12 treated with CK for 60 min. Scale bars 10 µm and 0.5 µm, respectively. G. CMT plus-end growth rates (µm/min) quantified from the EB1b-GFP trajectories tracked during 5 min in epidermal cells of the EZ treated with mock (DMSO) or CK. In the boxplots, the center lines show the 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. (ns = non-significant, *** P < 0.001 by Student's t-test, n= 7-9 cells with 7 roots per condition in 3 independent experiments). Concentrations and treatment conditions (F, G) were as for (A, B).

H, I. CMTs visualized with MAP4-GFP in root epidermal cells of the cre1-12 mutant. Cells at the TZ, the EZ and the DZ were monitored at time point 0 and 60 min after treatment with oryzalin (1 µM) (H), and CK plus oryzalin (I). As CK source 10 µM BAP was used and 1 µM oryzalin. For double (CK and oryzalin) treatment roots were pretreated for 60 min with CK and then transferred to medium supplemented with both compounds. Scale bar 10 µm.

J. Quantification of the MAP4-GFP CMTs reporter signal in cre1-12 root epidermal cells at different root zones treated with mock (DMSO, white box), oryzalin (1 µM, light grey box) and CK (10 µM BAP) plus 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 < 0.0001 by Student's t-test, n= 3-10 cells per root growth zone with 5-8 roots per condition in 4 independent replicates).