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Imaging Rhizosphere pH and CO2 Dynamics

Plant Root - Soil Interactions Quantified with Prototype VisiSens Systems

S. Blossfeld1, C. M. Schreiber2, G. Liebsch3, A. J. Kuhn1, and P. Hinsinger2
1Forschungszentrum Jülich GmbH, Institute of Bio- and Geosciences, IBG-2: Plant sciences, Jülich, Germany
2INRA, UMR Eco&Sol, Montpellier, France
3PreSens Precision Sensing GmbH, Regensburg, Germany

Prototype VisiSens™ devices for pH & CO2 imaging were used to perform continuous and highly resolved measurements in rhizobox systems. pH was measured around durum wheat roots intercropped with chickpea roots, while CO2 dynamics were recorded in the rhizosphere of Viminaria juncea. The 2-dimensional analyte maps revealed that wheat root tips acidified slightly, while the root hair zone basified the rhizosphere. Chickpea roots strongly acidified the surrounding area, except for regions where wheat roots crossed, which remained more alkaline. CO2 measurements showed that growing root of Vininaria juncea had influence on soil CO2 content in its surrrounding. Non-invasive imaging with planar optrodes is an ideal method in the heterogeneous environment of root research applications.

The region of soil directly influenced by roots and associated microorganisms is called the rhizosphere. Investigating biochemical processes in this highly heterogeneous and non-sterile environment is very difficult and often limited by the available monitoring technology. In this study chemical optical sensor foils have been applied to record pH and CO2 dynamics around growing roots of different plant species. The prototype pH and CO2 imaging devices (VisiSens™ A2 and A3, PreSens) allow non-invasive measurement of these key rhizosphere parameters, so close-to-natural set-ups can be investigated. Therefore, chemical optical sensor foils were mounted in rhizoboxes and 2-dimensional analyte maps recorded with the VisiSens™ fluorescence microscopes.

Materials & Methods

Durum wheat (Triticum turgidum durum 'LA 1823') and chickpea (Cicer arietium 'ILC 01302', not inoculated with Mesorhizobium ciceri) were grown under greenhouse conditions (25 ± 2 °C, 8/16 h dark/light cycles, 50 - 55 % rel. humidity). Two weeks after germination both species were planted together in one rhizobox (40 x 20 x 2 cm) to investigate root interactions between species. The rhizobox was kept in a climate chamber (20 °C, 10/14 h dark/light cycle, 200 µmol photons m-2 s-1, 60 % rel. humidity). It had a single front plate, which had to be removed to install the sensor foils (Fig. 1 A). During root growth areas of interest were chosen in the root systems, i. e. the crossing over of roots of both species to monitor spatial and temporal dynamics of pH changes. After sensor foil installation the rhizoboxes were tilted to 30° from the vertical, to force root growth right behind the sensor foil, and the camera was fixed 60 mm from the sensor foils to monitor pH dynamics at 10 min. intervals over the next days. Viminaria juncea was grown in a climate chamber (20 °C, 10/14 h dark/light cycle, 200 µmol photons m-2 s-1, 60 % rel. humidity). For the experiments it was planted and grown in a waterlogged sand-filled rhizobox (40 x 20 x 2 cm). Soil temperature was kept at constant 20 °C to maintain equal respiration rates of roots and microorgan- isms throughout the experiments. The rhizobox had to be opened and CO2 sensor foils were placed onto the front plate of the box (Fig. 1 B). After closing the rhizobox was tilted at 30 ° from the vertical. Then the rhizobox was set to waterlogged conditions with nutrient solution again. After 1 d of equilibration, the VisiSens A3 detector unit was fixes onto the front plate opposite the sensor foil and CO2 content of the area was measured at 10 min intervals over the next 3 days.

pH around Intercropped Wheat and Chickpea Roots

We monitored pH dynamics in the rhizobox with both species - durum wheat and chickpea - growing together for 10 days using the prototype VisiSens A2. The maps showed pH changes around developing lateral roots of chickpea and a growing wheat root. The tip of the growing wheat root showed a zone of acidification (from pH 8 to pH 7) along the elongation zone. This acidification stopped, when the elongation zone passed by and root hairs were formed (Fig. 2, upper left). The soil gets alkalized probably due to the large nitrate uptake of the roots. The excess anion uptake is compensated by proton influx. With the chemical optical sensors the slight acidification at growing root tips could also be detected, which might have been unnoticed with other measurement methods. Here a greater uptake of anions than cations takes place, which is in accordance with the acid growth mechanism described in literature. In the center of our recorded maps a chickpea root grew from top to the bottom, and developed two lateral roots during the investigated period. The main root and the basal parts of the lateral roots acidified their rhizosphere strongly down to pH 6 or lower (Fig. 2). Only the area where the wheat root crossed the chickpea root, in the upper left corner, the rhizosphere remained more alkaline. The tip of the upper lateral chickpea root showed a clearly localized zone of acidification moving through the soil over time as the root grew.

CO2 around Viminaria juncea Roots

A growing root tip of V. juncea showed increased pCO2 levels during a three day monitoring with the prototype VisiSens A3 system (Fig. 3). pCO2 changed along with the growing root and reached values of up to 70 % CO2. This strong impact on soil pCO2 was detected in an area several millimeters around the growing root. The changes in pCO2 are probably not only caused by autotrophic respiratory activity but also by heterotrophic activity of a biofilm on the root surface. The waterlogged conditions cause a rise in CO2 due to the reduced diffusion of CO2 in water. The sensor foils proved to be an appropriate tool to measure CO2 in soil at the spatial scale of hotspots of biological activity.

Conclusion

With the new prototype systems we were able to monitor pH changes in very heterogeneous and dynamic microenvironments and even detected opposite behavior for roots of different plant species. The CO2 sensor foils gave us the opportunity to make completely new findings. Generating quantitative maps of key rhizosphere parameters with high spatial resolution is a great advantage. The VisiSens systems proved to be a major step forward when investigating root - soil interactions. As the systems are fairly easy to handle and soon there might be the possibility to measure even more analytes these devices might be established in root research.

Application note adapted from
Blossfeld, et al.: Quantitative imaging of rhizosphere pH and CO2 dynamics with planar optodes, Annals of Botany (2013) 112 (2), 267 - 276

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