Tissue preparation for microscopy

2.10.1 Clearing roots for DIC microscopy

Roots were cleared as described in Malamy and Benfey (1997). Briefly, samples were incubated in a small petri dish containing 0.24 N HCl and 20% methanol at 60o C for 30 min. Samples were transferred to a solution of 7% NaOH in 60% ethanol for 15 min at room temperature. Roots were rehydrated by incubating in 40% ethanol, 20% ethanol and 10% ethanol solutions for 5 min each. Samples were infiltrated for 15 min in 5% ethanol, 25% glycerol. Roots were mounted on a microscope slide in 50% glycerol.

2.10.2 Confocal microscopy

To visualise cell walls, roots were stained with propidium iodide (PI; P4170; Sigma Aldrich). Whole seedlings were submerged in 100 µg/ ml PI in water for 2 min and 30 sec. Samples were mounted in water on a slide under a coverslip for imaging. A Zeiss

|40|

LSM780-UV-NLO confocal laser scanning microscope was used. Images were acquired and analysis using Zen 2012 digital imaging software (Zeiss).

2.11 Flow cytometry

Samples were prepared based on the method described by Galbraith et al. (2001) and Dolezel et al. (2007). Root tips from at least 300 seedlings were excised using a razor blade and chopped in approx. 1 ml ice cold Galbraith’s buffer (45 mM MgCl2, 20 mM

MOPS, 30 mM sodium citrate, 0.1% (vol/vol) Triton X-100.) for no more than 2 min. Samples were mixed by pipetting and passed through a 50 micron nylon mesh and centrifuged for 10-15 min at 150 g. The supernatant was removed, resulting in approx. 200 µl of remaining liquid. Samples were stored on ice for up to 4 h. Approximately 20 min before analysis, 100 µg/ml PI (P4170; Sigma Aldrich) was added and samples were vortexed. Samples were vortexed again prior to and during flow cytometry. Nuclei were analysed with a BD LSRFORTESSA flow cytometer (BD Biosciences) at the John Curtain Medical School Cytometry and Imaging Facility. Cell cycle analysis was performed with ModFitLT software (Verity Software House).

2.12 DR5:Luc assays

DR5 is a synthetic auxin responsive element used to monitor endogenous auxin levels (Ulmasov et al., 1997). The method for DR5:Luciferase (Luc) assays and imaging were based on methods described by Moreno-Risueno et al. (2010) but optimised for the equipment available at the ANU Research School of Biology as follows. Five mM potassium luciferin (LUCK-100; Gold Biotechnology) was pipetted onto seedling 10-15 min prior imaging. Ten images with 30 second exposures were taken with a cooled Andor DV425-BV camera. The median of the 10 images was obtained using ImageJ software, which gave reproducible results (Fig. 1).

|41|

Figure 1. Optimised images of a seedlings containing DR5:Luc construct. Seedlings were grown vertically on ½ MS medium with 0.8% agar and 24 h light. Five mM potassium luciferin was pipetted onto the seedling 10-15 min prior imaging. 10 images with 30 second exposures were taken with a cooled Andor DV425-BV camera. The median of the 10 images was obtained using ImageJ software.

This method allowed the visualization of static luminescent points, as opposed to the oscillatory pulsing observed only at the root tip described by Moreno-Risueno et al. (2010). In order to validate that these points did in fact mark the site of future lateral roots, several experiments were undertaken in consultation with members from the Benfey Lab. Firstly, the periodicity of luminescent point establishment was examined by performing a time course (Fig. 2). Results showed that on average, the periodicity of luminescent points was slightly higher (one every 8 h) than the 6 h reported by

Moreno-Risueno et al. (2010).

Figure 2. Images of a seedlings containing DR5:Luc construct at specified days post imbibition (dpi). Red arrows indicate the root tip, green line indicates root shoot junction.

|42|

Secondly, after initially imaging luminescent points on 6 day old plants, the root tips were cut off (to encourage lateral root emergence) and plants were left to grow for 3 days before being imaged again. To examine whether LRs emerged from luminescent points, luminescent images from 6 dpi plants were overlayed on brightfield images from day 9 (Fig. 3). This revealed that the vast majority of LRs emerged only from luminescent points. On occasions, LRs did not develop from luminescent points, and more rarely, LRs emerged from points where there was no luminescence. It was therefore concluded that luminescent points were indicative of sites that had the potential to become lateral roots.

Figure 3. Chemiluminescent images taken at 6 dpi overlayed on brightfield images taken at 9 dpi.

The methods developed here were subsequently validated in a publication by Van Norman et al. (2014), who described the luminescent points as a measure “lateral root competency”.

2.13 EdU assays

EdU (5-ethynyl-2'-deoxyuridine) assays were performed to visualise cells in the root tip undergoing DNA synthesis (a hallmark of the S phase of the cell cycle). EdU staining and visualisation was performed as described in Kotogany et al. (2010). Briefly, 1 µM EdU (in appropriate MS medium) was added to wells/plates and incubated for 30 min. Samples were fixed with 4% (w/v) formaldehyde solution in phosphate-buffered saline (PBS) with 0.1% Triton X-100 for 30 min and washed 3 times with PBS. Alexa-Fluor-488 azide was coupled to the EdU alkyne using the Click-It EdU Alexa Fluor 488 Imaging Kit (10337; ThermoFisher Scientific) according to manufacturer’s instructions. Briefly,

|43|

samples were incubated with the Reaction Cocktail in the dark for 30 min before being washed 3 times with PBS. Samples were mounted in water and observed with a Leica DM5500 microscope.

2.14 References

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method forAgrobacterium-

mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735- 743.

Dolezel, J., Greilhuber, J., and Suda, J. (2007). Estimation of nuclear DNA content in

plants using flow cytometry. Nature Protocols 2, 2233-2244.

Galbraith, D.W., Lambert, G.M., Macas, J., and Dolezel, J. (2001). Analysis of nuclear

DNA content and ploidy in higher plants. In Current Protocols in Cytometry (John Wiley & Sons, Inc.).

Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAY™ vectors for Agrobacterium-

mediated plant transformation. Trends in Plant Science 7, 193-195.

Kotogany, E., Dudits, D., Horvath, G., and Ayaydin, F. (2010). A rapid and robust assay

for detection of S-phase cell cycle progression in plant cells and tissues by using ethynyl deoxyuridine. Plant Methods 6, 5.

Lobet, G., Pagès, L., and Draye, X. (2011). A novel image-analysis toolbox enabling

quantitative analysis of root system architecture. Plant Physiology 157, 29-39.

Malamy, J.E., and Benfey, P.N. (1997). Organization and cell differentiation in lateral

roots of Arabidopsis thaliana. Development 124, 33-44.

Moreno-Risueno, M.A., Van Norman, J.M., Moreno, A., Zhang, J., Ahnert, S.E., and Benfey, P.N. (2010). Oscillating gene expression determines competence for

periodic Arabidopsis root branching. Science 329, 1306-1311.

Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays

with tobacco tissue cultures. Physiologia Plantarum 15, 473-497.

Van Norman, J.M., Zhang, J., Cazzonelli, C.I., Pogson, B.J., Harrison, P.J., Bugg, T.D.H., Chan, K.X., Thompson, A.J., and Benfey, P.N. (2014). Periodic root branching in Arabidopsis requires synthesis of an uncharacterized carotenoid derivative.

|44|