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.