In Vivo Imaging and Quantification of Carbon Tracer Dynamics in Nodulated Root Systems of Pea Plants

Abstract

Legumes associate with root colonizing rhizobia that provide fixed nitrogen to its plant host in exchange for recently fixed carbon. There is a lack of understanding of how individual plants modulate carbon allocation to a nodulated root system as a dynamic response to abiotic stimuli. One reason is that most approaches are based on destructive sampling, making quantification of localised carbon allocation dynamics in the root system difficult. We established an experimental workflow for routinely using non-invasive Positron Emission Tomography (PET) to follow the allocation of leaf-supplied ¹¹C tracer towards individual nodules in a three-dimensional (3D) root system of pea (Pisum sativum). Nitrate was used for triggering a reduction of biological nitrogen fixation (BNF), which was expected to rapidly affect carbon allocation dynamics in the root-nodule system. The nitrate treatment led to a decrease in ¹¹C tracer allocation to nodules by 40% to 47% in 5 treated plants while the variation in control plants was less than 11%. The established experimental pipeline enabled for the first time that several plants could consistently be labelled and measured using ¹¹C tracers in a PET approach to quantify C-allocation to individual nodules following a BNF reduction. Our study demonstrates the strength of using ¹¹C tracers in a PET approach for non-invasive quantification of dynamic carbon allocation in several growing plants over several days. A major advantage of the approach is the possibility to investigate carbon dynamics in small regions of interest in a 3D system such as nodules in comparison to whole plant development.

Keywords: 11C; PET; biological nitrogen fixation; legumes; nitrate inhibition; radiotracer.

Figure 1

Flowchart of ¹¹C-PET experimental and data analysis pipeline.

Figure 2

Plant cultivation and labelling set-up. (A) Sixteen-days-old Garden pea (Pisum sativum L. cv. Kayanne) in PVC pots with nutrient solution. (B) Transparent stabilizing support grid for reducing root displacement during plant transfer between PET and MRI measurements. Arrow indicates region shown enlarged in panel (D). (C) Overview image of a pea plant with ¹¹CO₂ tracer labelling cuvette situated above the PET detectors positioned at the level of the pot. (D) Representative nodules in the root system of a pea plant. Scale bars, (A–C) 20 mm, (D) 2 mm.

Figure 3

Root system of a pea plant visualized by Magnetic resonance image (MRI) and Positron Emission Tomography (PET) with corresponding time series of ¹¹C tracer in one nodule ROI. (A) MRI shows the structure of a nodulated root system 16 days after transfer to the pots (DAT, see Methods). The arrow indicates an individual nodule for which data are shown in (C,D). Scale bar, 10 mm. (B) PET image of ¹¹C tracer distribution within the nodulated root system 13 DAT (maximum intensity projection). Color scale 0.0 (black)–1.0 (white) a.u. The arrow indicates the same nodule as in (A). (C) Time series of decay corrected ¹¹C radioactivity in the region of interest (ROI) of the nodule marked by the arrow in B for all eight consecutive measurements, i.e., two measurements per day on 13, 14, 15, and 16 DAT. (D) Time series of radioactivity in the same ROI normalized to the total radioactivity detected in the root system. Note that 15 DAT 07:59 is partly covered by almost identical curve of 14 DAT 10:55. The grey box indicates the data points used to calculate mean tracer fractions for each measurement of this ROI.

Figure 4

Example for carbon tracer allocation to whole root system and selected nodule ROIs of a pea (Pisum sativum L.) plant. (A) Two-dimensional projection of carbon tracer distribution within the nodulated root system obtained from PET data, overlaid with positions of 6 ROIs (numbered 1 to 6) enclosing selected nodules. Scale bar, 10 mm. Projections with ROIs from all spatial directions can be found in Figure S4. (B) Mean tracer fractions in ROIs from (A) (corresponding color) compared to total tracer detected in the root system, determined according to Figure 3D on four days with two measurements per day. Standard deviation (SD) was lower than marker size. (C) Radioactivity measured by a shoot and a root scintillation detector for the same timespan (75–105 min) as used for the calculation of the tracer fractions. Units are counts per second (cps), decay corrected mean ± SD. Whole plant shoot CO₂ uptake rate (mean ± SD) was determined over the full measurement period of 2 h. Note that while numbers for root and shoot detector appear to be similar, the radioactivity was much higher in the shoot, where a less sensitive detector was used to prevent excessive dead time effects. DAT, days after transfer to pots.

Figure 5

Carbon tracer allocation to nodules as fractions of the total tracer allocated to the root system detected by PET. (A) Two control plants (Ctrl P1 and P2). (B) Five plants treated with nitrate (Treat P1-5) in the root zone. Fractions for all six nodule ROIs per plant and labelling experiment were summed up to one data point. Standard deviation (SD) was lower than marker size. Individual ROI values for control Plant 1 are displayed in Figure 4, individual ROI values of the other plants can be found in Figure S2. The starting time of the treatment is indicated by a dotted magenta line in (B). DAT, days after transfer to pot.