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

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.


Background
Phytohormone auxin induces rapid growth in Arabidopsis thaliana hypocotyls. This process requires the TIR1/AFB-Aux/IAA auxin co-receptor. Auxin promotes the binding of TIR1/AFB and Aux/IAA, which leads to ubiquitination and degradation of the latter, and results in transcription of auxin-responsive genes. This protocol focuses on measuring the growth, auxin signaling and cell wall acidification in Arabidopsis thaliana etiolated hypocotyls. This protocol is based on the previous work of Schenck et al., 2010, Takahashi et al., 2012, Fraas et al., 2014and Spartz et al., 2014; but unlike the published work, we describe the procedures that enable measuring a larger spectrum of processes occurring during growth of hypocotyls; from the macroscopically visible organ elongation, cell wall pH monitored by confocal microscopy to the real-time nuclear auxin signaling visualized by luciferase bioluminescence.

2.
Prepare a depletion plate with 5 ml depletion medium (DM, Recipe 3) in a Falcon 60 x 15 mm dish. After solidification, place cellophane foil onto the surface. Damp the cellophane foil with liquid depletion medium solution.

3.
Place a dissecting microscope in a dark room and cover the illumination with a green filter made of 8 layers of green office foil ( Figure 1).

4.
Uncover the Petri dishes with seedlings and select the seedlings with similar hypocotyl length excluding the longest and shortest ones. Decapitate the seedlings right below the apical hook and before the shoot-root junction to get a hypocotyl segment by cutting them on the surface of the agar using a very sharp razor blade. Prepare 6-8 segments for each treatment. Using sharp forceps, transfer the segments onto the cellophane foil in the depletion plate without squeezing them, the sample preparation procedure is depicted in Figure 2. Keep in darkness for 30-60 min.

5.
Afterwards, transfer the segments by flipping the cellophane foil onto a treatment plate with the depletion medium supplemented with the desired treatment ( Figure 2), in our case 10 µM 3-Indoleacetic acid (IAA) and the mock control (Ethanol equivalent).

6.
Immediately place the treatment plates on a flatbed scanner, imaging through the layer of the phytagel. A wet black filter paper is placed into the lid of the dish to improve the contrast of the image. Scan the samples in the 8-bit grayscale and at 2,400 dpi every 10 min automatically using the AutoIt program (see Supplemental file 1).

5.
Using a confocal microscope with a 20x/0.8 Plan-Apochromat M27 objective, set the position of each segment using the position manager so that the apical region of the hypocotyl segment is imaged. Image 5 zsections, z-thickness matched to the pinhole size, of each hypocotyl segment.

6.
Set the microscope for simultaneous imaging of GFP and RFP by exciting using 488 and 555 nm diode lasers, and splitting the emitted light with a short pass 550 nm and long pass 560 nm filters, 16 bits per pixel. Image all positions every 5 min.

A.
Hypocotyl elongation image analysis

1.
To achieve unbiased measurement, we created a Fiji macro (see Supplemental file 1) for analyzing the length of the segment at each time point. The macro firstly creates the time lapse of the image sequence captured from the scanner, then allows you to manually create a rectangle ROI for each segment, followed by automatically thresholding each ROI and measuring the Feret's diameter, the maximum caliper, as the length of the segment. The macro eventually generates '.txt' file for each ROI or hypocotyl, including the Feret's diameter of that hypocotyl in each time point.

2.
Copy and paste the result into Excel, set the initial length of the segment as 100%, and calculate the length of the hypocotyl at each time point to obtain a growth curve ( Figure 4C). Besides, growth can be visualized by creating a montage-kymograph of individual hypocotyl segment in Fiji ( Figure 4A).

B.
Analysis of the bioluminescence intensity

1.
Analyze the image sequence in Fiji (Schindelin et al., 2012). Manually outline all the segments via Polygon selection, at their brightest time frame, and add them into the region of interest (ROI) manager, followed by multi-measuring the mean grey value. This gives the average intensity of each segment at each time point.

2.
Copy and paste the result into Excel. Take the initial intensity of the segment as 100%, and analyze the average of the luminescence intensity of each hypocotyl at each time point, to get an intensity curve in time. Additionally, one can visualize the growth and the luminescence intensity by creating a montage-kymograph in Fiji ( Figures 4B and 4D).

C.
Image analysis of the cell wall pH

1.
Analyze the apoplastic pH using Fiji. We use the SUM projections of the z-stacks ( Figure 5A). Set the threshold of the apoplast region using the RFP channel so that only the cell wall signal is selected. Create the selection using the 'create selection' command and measure intensity in GFP and RFP channels. Analyze the intensity ratios in Excel program. We analyze the apoplastic pH change in relative values the lower the GFP intensity, the lower the apoplastic pH is (Gjetting et al., 2012).

2.
Alternatively to Step C1, the apoplastic pH can be visualized and measured using the AreaKymo MATLAB ® script, Figure 5B (Supplemental file 1). The AreaKymo script essentially does the same procedure as described in Step C1, but does so automatically without the user input, allowing for rapid processing of several hypocotyls at a time. The user first merges several hypocotyl SUM projection time series into one 'tif.' series using the Fiji program ('combine stacks' command) and converts them into 16-bit 'tif.' images (MATLAB does not handle well the 32-bit images that the SUM projection command creates). Then the user should find a threshold of the RFP channel that is optimal for selecting just the apoplast signal. In MATLAB, the AreaKymo script is run, the combined time series is selected, and the user specifies the value of the threshold for the RFP channel and the desired width of the rectangle that will represent the individual timeframe. The script outputs the visual representation of apoplastic pH and also the values in the form of a series of boxplots.

Notes
During all steps where the hypocotyls are manipulated (cut and transferred to new plates) it is crucial to be extremely gentle with the tissue, not squeeze it but rather scoop it using sharp forceps. The tissue needs to be protected from drying; plates must be kept closed whenever possible to prevent excessive evaporation.

1.
Chlorine   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. 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.