200µm Maize Root Sections cut using 7000smz-2 vibrotome

Abstract

(from https://www.biorxiv.org/content/10.1101/2021.01.15.426842v1)

Radial expansion is a classic response of roots to mechanical impedance that has generally been assumed to aid penetration. We analysed the response of maize nodal roots to impedance to test the hypothesis that radial expansion is not related to the ability of roots to cross a compacted soil layer. Genotypes varied in their ability to cross the compacted layer, and those with a steeper approach to the compacted layer or less radial expansion in the compacted layer were more likely to cross the layer and achieve greater depth. Root radial expansion was due to cortical cell size expansion, while cortical cell file number remained constant. Genotypes and nodal root classes that exhibited radial expansion upon encountering the compacted soil layer also thickened in response to exogenous ethylene in hydroponic culture, i.e. radial expansion in response to ethylene was correlated with the thickening response to impedance in soil. We propose that ethylene insensitive roots, i.e. those that do not thicken and are able to overcome impedance, have a competitive advantage under mechanically impeded conditions as they can maintain their elongation rates. We suggest that prolonged exposure to ethylene could function as a stop signal for axial root growth.

Method: Immediately after scanning, all soil columns were lifted out of the plastic columns and roots were washed from the soil. The entire root system was extracted and stored in 75% ethanol (v/v) until sectioning. Penetrating roots of node 3 and node 4 were selected for sectioning based on polylining results and clipped from the entire root system. The length along each individual root axis was measured and sectioning positions were identified along the root axis of interest. Pieces of root containing the sectioning positions were excised out of the root axis and embedded by placing them into 3D printed moulds. 6% agarose (Sigma-Aldrich Co. Ltd, Gillingham, UK) at 39°C was used to fix the roots within the mould. A vibrating microtome (7000 smz-2) (Campden Instruments Ltd., Loughborough, UK) was used to section the roots within the agarose block at 200-230 µm thickness per slice (blade speed at 1.75-2 mm/s, blade frequency at 70 µm). Root sections were then incubated in calcofluor white (Sigma-Aldrich, Co. Lt, Gillingham, UK), 0.3 mg/ml for 90 seconds, rinsed with deionised water and placed on a microscopy slide and covered by a coverslip.