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.