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. 2018 Jan 5;8(1):e2685.
doi: 10.21769/BioProtoc.2685.

Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls

Lanxin Li  1 , S F Gabriel Krens  1 , Matyáš Fendrych  1 , Jiří Friml  1
Affiliations

Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls

Lanxin Li et al. Bio Protoc. .

Abstract

The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.

Keywords: Auxin signaling; Cell elongation; Cell wall pH; Hypocotyl; Live-cell imaging.

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Figures

Figure 1.
Figure 1.. Binocular dissecting microscope with a green filter
Figure 2.
Figure 2.. The sample preparation procedure
Figure 3.
Figure 3.. Dark box with a Photometric Evolve EMCCD camera
Figure 4.
Figure 4.. Hypocotyl segment growth and DR5::LUC intensity measurements.
A. The kymograph of the hypocotyl segment of Col-0 under mock treatment (upper row) and 10 μM IAA treatment (lower row) from 0 to 460 min; time interval of 10 min. The kymograph was done by making montage of the growing part of one representative sample in Fiji. (Note that to visualize the growth better, the upper half part of the hypocotyl where growth takes place was used for making montage). Vertical and horizontal scale bars represent 1 mm and 20 min, respectively. B. The kymograph of the luminescence intensity in the mock-treated DR5::LUC hypocotyls (upper row) and 10 μM IAA-treated (lower row) from 0 to 172 min; time interval of 2 min. The ‘FIRE’ look-up table was applied in Fiji. Scale bar is 10 min. C. Quantification of the growth of Col-0 hypocotyls treated with mock or 10 μM IAA from 0 to 460 min. The growth is expressed as the percentage of the original segment length. D. Quantification of the luminescence intensity in the DR5::LUC hypocotyls treated with mock or 10 μM IAA. The Fold change is the average intensity of the hypocotyls normalized by the intensity at timepoint 0.
Figure 5.
Figure 5.. Analysis of the apoplast pH.
A. The apoplast pH after application of auxin to a hypocotyl expressing the apo-pHusion sensor. Auxin application is indicated with an arrowhead, GFP is shown in green while RFP in magenta. B. The output of the AreaKymo MATLAB® script. Time is progressing from top to bottom; ratio of the two fluorophores is shown using the ‘FIRE’ look-up-table.
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