Radial Oxygen Loss from Plant Roots-Methods

Abstract

In flooded soils, an efficient internal aeration system is essential for root growth and plant survival. Roots of many wetland species form barriers to restrict radial O₂ loss (ROL) to the rhizosphere. The formation of such barriers greatly enhances longitudinal O₂ diffusion from basal parts towards the root tip, and the barrier also impedes the entry of phytotoxic compounds produced in flooded soils into the root. Nevertheless, ROL from roots is an important source of O₂ for rhizosphere oxygenation and the oxidation of toxic compounds. In this paper, we review the methodological aspects for the most widely used techniques for the qualitative visualization and quantitative determination of ROL from roots. Detailed methodological approaches, practical set-ups and examples of ROL from roots with or without barriers to ROL are included. This paper provides practical knowledge relevant to several disciplines, including plant-soil interactions, biogeochemistry and eco-physiological aspects of roots and soil biota.

Keywords: methylene blue staining; microelectrodes; microsensors; planar optodes; root-sleeving electrodes.

Figure 1

Radial O₂ loss from individual adventitious roots of rice grown in aerated or stagnant, deoxygenated conditions. In aerated nutrient solution, rice does not form an ROL barrier (with the exception of some wild accessions [20]), whereas the barrier is formed in stagnant, deoxygenated nutrient solution. Points indicate mean radial O₂ loss at different distances behind the root tip and bands represent standard error (figure was constructed compiling published information on ROL from rice roots [11,12,13,14,15,16,17,18,19,20], n = 28). Plants were grown in aerated or stagnant, deoxygenated conditions for 5 to 31 days. Lengths of the roots were 93 to 159 mm.

Figure 2

Colorimetric methylene blue method for root radial O₂ loss determination. (A) General set-up including photo chamber filled with deoxygenated solution and reduced methylene blue dye. Intact rice plants are inserted and fixed to the photo chamber. Light panels in A are provided to ensure photosynthesis and internal O₂ transport and for enhanced photo collection. (B) Rice roots with no barriers to ROL. (C) Rice roots with barriers to ROL. (D) Sites of O₂ loss through root ‘windows’ of developing laterals (arrows). Rice plants in A and B were grown for 28 days in aerated or stagnant, deoxygenated solutions, respectively. Rice plants in D were grown for one week in aerated solutions with high concentration of Fe (300 µM). Arrowheads in B and C point to blue halos where O₂ loss is occurring.

Figure 3

Polarographic method for radial O₂ loss determination in roots. (A) General set-up including a transparent acrylic chamber filled with deoxygenated solution and the roots of an intact rice plant submerged. Different electrodes (see text for details) are inserted into the deoxygenated solution and connected to a polarograph. (B) Current–voltage curve for O₂ determination in solutions. (C) Close-up of a root inserted into a Pt-sleeving electrode. (D) Diffusion current from the Pt-sleeving electrode. Arrowhead in B points to current plateau where the rate of O₂ reduction is independent of voltage and only depends on the rate of O₂ diffusion to the cylindrical electrode surface. Arrows in D indicate the points when Pt-sleeving electrode was moved in 10 mm steps from basal parts toward the tip of a root of Hordeum marinum grown in aerated solutions. Note the time taken for current stabilization before a new measurement.

Figure 4

O₂ microelectrodes and O₂ micro-optodes for ROL determination from roots. (A) Clark-type O₂ microelectrode with guard cathode. (B) O₂ micro-optode. (C) General set-up including a transparent acrylic chamber filled with deoxygenated solution and the submerged roots (detached) of a rice plant. The O₂ microelectrode (to obtain spatial concentration profiles) and the O₂ micro-optode (to follow the O₂ in the bulk) are mounted in a motorized stage and connected to a picoammeter and an optode meter, respectively. (D) Close-up of an O₂ microelectrode near the root surface of Zea nicaraguensis. (E) Characteristic O₂ gradients from roots without a barrier (roots from Zea nicaraguensis plants grown in aerated nutrient solutions for 25 days, left) and a tight barrier to ROL (roots from Zea nicaraguensis plants grown in staganant, deoxygenated nutrient solutions for 25 days, right). Arrow in C points to O₂ microelectrode, arrowhead to temperature sensor and asterisk key to O₂ micro-optode. Arrow in D shows the tip of an O₂ microelectrode.

Figure 5

Planar optodes for ROL determination from roots. (A) General set-up, including a chamber filled with anoxic medium and the roots of an intact rice plant submerged, a digital camera, blue LED lights and software for image analysis (see text and Supplementary Information S4 for details). (B) Image collection process in darkness. (C) O₂ planar optode image indicating differences in ROL in roots of Puccinellia festuciformis grown in aerated conditions for 5 weeks. (D) ROL quantification in the regions of interest. Oxygen concentrations in (D) refer to specific positions of the root, as shown in (C).