Materials and methods

Plant material

Strawberries (Fragaria × ananassa Duch. cv. Sonata) were planted in substrate-filled plastic pots (1-L volume). They were grown on a cultivation bed in a commercial greenhouse located in Meerle (Hoogstraten, 51°27’12.8”N 4°47’40.8”E). Inside the greenhouse, air temperature was maintained at 18/8 °C (day/night) with a central heating system. Light intensity was maintained above 89 µmol m-2 s-1 for nine hours during the daytime by supplemental lighting. CO2 concentration was maintained at 800 ppm while relative humidity was set at 68/85 % (day/night). Plants were watered from two hours after lights were switched on, until two hours after sunset. Flowers were pollinated by bumblebees. After fruiting, the plants were translocated to the INFINITY lab of Ghent University (Belgium) at Ghent University Hospital and analysed by a small animal PET scanner. In total three plants were analysed, each having bunches with fruits (scanned bunches in this study contained six or eight fruits) with different development stages defined by colour (green colour: first stage at which the fruit is just fully formed; white colour: second stage at which the fruit begins to rapidly expand and becomes much larger; red colour: third and final stage at which the fruit is mature) and size. In addition, two plants had large petiole length and one had smaller petiole length. The average distance between source leaf and each fruit (petiole + peduncle) of the large plants was about 14 % higher than that of the smaller plant (50.5 ± 1.2 cm vs. 44.2 ± 0.4 cm). In this study, we grouped the analysed fruits by the following code: colour - average fruit size (mm) - plant size (Large or Small), because the plant size was expected to affect the translocation rate.

11CO2tracer production

11CO2 was produced by using a cyclotron (18 MeV Cyclone, IBA, Ghent Belgium) at UZ Ghent (see Minckeet al. (2018) for detailed information). Inside the cyclotron,11C-radioisotope was produced through the14N(p, α) 11C reaction by bombarding N2/H2 (5%) target with an energetic proton beam resulting in 11CH4. This11CH4 was cryogenically concentrated and released by heating. Subsequently, it was pushed out by a small flow of helium through a heated reactor tube with cobalt oxide. As such, it was oxidised to 11CO2 which was finally captured in basic NaOH by bubbling it through a 1 M solution of NaOH. The average starting activity was 732 ± 41 MBq (SE, N = 10). Note that the 11CO2 concentration is negligible with regard to the total CO2 concentration in the atmosphere since it corresponds to 2.14 ± 0.12 pmol11CO2.

Setup of PET measurement

In this study, a LabPET8 scanner (TriFoil Imaging, Chatsworth, CA, USA) was used, located at INFINITY lab. Detectors consisted of LGSO (Lu0.4Gd1.6SiO5:Ce) and LYSO (Lu1.9Y0.1SiO5:Ce) scintillation crystals and avalanche photodiodes were used for read-out. The detection ring had a diameter of 15 cm and a depth of 7.5 cm. Twice a year, the detectors are normalised, and the scanner is calibrated. Further detailed information on the LabPET8 is described in Hubeauet al. (2018).
To analyse photosynthate translocation dynamics, the strawberry plant was placed in front of the PET scanner while the fruit bunch, which had fruits at various developmental stages, was put inside the detection ring (i.e. field of view - FOV). The fruits were positioned on foam plastic to put them as close as possible to the centre of the FOV (Fig. 1). The leaf developing immediately below the fruit bunch was inserted into an “exposure bag” (see Hubeau et al. (2018) for detailed information), i.e. a clear plastic bag for feeding11CO2 gas to the leaf. To prevent leakage of the fed 11CO2 gas, the exposure bag was sealed at the entry point of the leaf petiole by a small 3-cm-long piece of plastic tubing which was cut longitudinally and covered on the inside with vacuum grease (Dow Corning, Auburn, MI, USA). Then the exposure bag was completely closed with cable ties around the tubed piece. To supply fresh air to the source leaf, the exposure bag was connected to an air circulation system which consisted of a gas analyser (model LI-7000, Li-Cor Inc., Lincoln, Nebraska, USA), a portable photosynthesis system (model LI-6400, Li-Cor Inc., Lincoln, Nebraska, USA), flow meters (AWM5101, Honeywell, Morris Plains, NJ, USA), a CO2 trap containing soda lime pellets (calcium hydroxide on sodium carbonate carrier, Merck, Overijse, Belgium) and tubing (inner diameter 4.0 mm and outer diameter 5.6 mm). The air inflow was controlled by the LI-6400 and supplied to the exposure bag at a rate of 400 mL min-1 and CO2 concentration of 400 ppm. For radiation safety, outflowing air was directed through a CO2 trap before being released into the atmosphere. In- and outflow passed through flow meters and the LI-7000. Flow meters monitored in- and outflowing flow rate to detect potential leakage of the exposure bag. LI-7000 measured water and CO2 content in the air entering and leaving the exposure bag to calculate transpiration and photosynthesis rates of the leaf. The exposure bag was also connected to a vial filled with 2 mL of H2SO4 through tubing (inner diameter 0.9 mm and outer diameter 1.5 mm) to allow the introduction of11CO2. The actual connection between the tube and the exposure bag was via a needle (inner diameter 0.5 mm) which was inserted through a septum. Dissolved11CO2 in NaOH arrived in a syringe, which was emptied in the vial at the start of the experiment. The NaOH got neutralised by the acid in the vial, releasing airborne11CO2. To strip as much11CO2 from the solution, air from the room was injected in the vial with a 50 mL syringe through a second needle (inner diameter 0.5 mm), which was submerged in the solution. The released 11CO2 gas was subsequently directed through a third wider needle (inner diameter 1 mm) positioned in the headspace of the vial which was, in turn, connected to the tube supplying the 11CO2 to the exposure bag. 11CO2 which was not assimilated by the leaf passed through a CO2 trap and was captured. Above the exposure bag, four 1.5-m-long LED arrays (Green Power LED production module deep red/blue, Philips, Amsterdam, The Netherlands) were placed. Light intensity at the leaf surface (inserted into the exposure bag) was adapted by changing height between the LEDs and the source leaf.

Procedures of PET experiment

Before the start of each PET measurement, air was fed to the exposure bag by the air circulation system. From the moment11CO2 was introduced into the exposure bag PET data was collected, and the air circulation system was stopped for 7 min so that the leaf could assimilate11CO2 well. Afterwards, the air circulation system was started again. After 120 min from11CO2 injection, the remaining radioactivity inside the H2SO4 vial was measured by a curie-meter to eventually calculate the injected radioactivity, which was used for normalisation of the data (see further). After 180 min, the PET measurement was stopped. Series of three or four PET measurements on the same source leaf were executed by changing light intensities at the leaf surface; i.e. 400 (N = 3), 200 (N = 2), 100 (N = 2), 50 µmol m-2 s-1(N = 3). Measuring longer than180 min is not recommended since then only 1/512 of the starting 11C-activity remained, i.e. approaching the sensitivity threshold of the PET scanner.

Translocation analysis

LabPET software version 1.12.1 (TriFoil Imaging, Chatsworth, CA, USA) was used to reconstruct the PET data with a temporal resolution of 10 min. The exponential decline in activity, due to decay of the radioactive isotope, is accounted for by the software during image reconstruction. Thereafter, PET images were obtained at 10 min intervals which were corrected for the radioactive decay. The resulting output was analysed using AMIDE v. 1.0.4-1 (Loening & Gambhir 2003). There, the dynamics of 11C-translocation into the fruits were computed by setting ellipsoid-shaped regions of interest (ROIs) around the fruits on the 3D PET images. To avoid ambiguity, we use the term “tracer” to mean “decay-corrected activity”, reserving the term activity for the detected events. From the ROIs, tracer profiles were generated showing the time course of the 11C-tracer intensities (in kBq) and will be referred to as time-tracer curves (TTCs). In total, 32 TTCs were obtained of 10 strawberry fruits (3 or 4 TTCs per fruit, which corresponds to the number of different light intensities that were applied on the source leaf). In this study, the amount of 11C-tracer per fruit was taken to represent real-time photosynthate accumulation. The photosynthate translocation rate into fruit under different light intensities was then calculated by the rate of tracer increase (slope of tracer profile) using the last 40 min of data. Constant flow could be assumed because a linear relationship was found during this period. Because the starting radioactivity differed between experiments, TTCs were normalised based on the amount of activity that was injected into the exposure bag. Furthermore, relative rate of photosynthate translocation into fruit was calculated by dividing the photosynthate translocation rate under the light intensity of 50, 100 and 200 µmol m-2s-1 by the photosynthate translocation rate under the light intensity of 400 µmol m-2 s-1. This normalisation was performed because photosynthesis was maximal at 400 µmol m-2 s-1, but differed for each of the plants under study. Finally, we obtained average relative rate of photosynthate translocation for each light intensity from all of the TTCs.

Photosynthetic light-response curve

After the PET measurement, the11CO2-fed leaf was taken out of the exposure bag and the photosynthetic light response curve (relationship between PPFD and photosynthetic rate) was measured using a portable photosynthesis system (model LI-6400, Li-Cor Inc., Lincoln, Nebraska, USA). Photosynthetic rates were measured under seven different light intensities, i.e., 1200, 800, 400, 200, 100, 50 and 0 µmol m-2 s-1 with an air temperature of 20°C, relative humidity of 50 % and CO2 concentration of 400 ppm in a leaf chamber. The air flow rate was maintained at 200 µmol s-1.