Measurement of p-coumaric acid in Pinus spp. pollen
To determine variation in p CA within pollen sporopollenin we used
thermally assisted hydrolysis and methylation pyrolysis-gas
chromatography and mass spectrometry (THM-py GC/MS). This method
developed by Blokker et al. (2005) was adapted by introducing an
internal standard where a solution of a known amount of nonadecanoic
acid was applied to each pollen sample during the THM reaction (Seddon
et al., 2017). Our sample preparation steps were as follows.
Pollen samples from one branch were submerged in distilled water in a
petri dish and three replicates with a predefined number of pollen
grains were then picked under a Leica DMIL 090-135.001 inverted
microscope with a self-customised Pasteur pipette. Pinus nigra, P.
pinaster and P. uncinata samples contained 150 pollen grains but due to
the smaller pollen size and in order to avoid THM-GC/MS results close to
the machine detection level, samples of P. sylvestris contained
200 pollen grains. The pollen samples were transferred to a microvial,
centrifuged and dried overnight at 50ºC. We then added 2 µl of a 25%
TMAH: Nonadecanoic acid: MEOH solution to the sample using a Hamilton
Digital 1701RN 10µl syringe. The samples were centrifuged and left in
room temperature for 30 minutes before they were placed in an oven at
70° for 2 hours. Measurements of p CA were made using an Hewlett
Packard (HP) 7890 Gas Chromatographer with an HP 6890 detector. The
pyrolysis unit is a GL Sciences ATAS-GL LINEX with a PAL Combi robotic
auto sampler. To check for analytical consistency between runs, we ran
samples with equal amounts of p CA and nonadecanoic acid after
every second pollen sample. For all experiments, The pyrolysis heating
programme was set to rise from 40 °C to 600 °C (maximum) at the maximum
ramp rate (approximately 60 °C/s). The GC oven was programmed from 40 °C
(6 minute hold time) to 130 °C at 15 °C/min followed by 250 °C at 8
°C/min, up to 320 °C at 15 °C/min and 1.5 min isothermal at 320 °C
(Willis et al., 2011).
We measured a total of 108 samples for p CA using TMH-py GC/MS. In
the cross-year study, we measured four species with one to three trees
per species and three replicates per tree, resulting in 48 analysed
samples. In the shading experiment we measured 60 samples; ten trees,
two treatments, and three replicates (Table 1). p CA ratios were
calculated by dividing the measured p CA peak area with measured
nonadecanoic acid peak area.