2.3 | Leaf sampling
For each species, we sampled foliage from three juvenile plants in each
of six or seven fenced plots and six or seven unfenced plots (six if
there were not seven with a sufficient number of plants). In an attempt
to limit variation in plant age, we selected individuals that were
within the middle 50% of the range of heights for that species in open
plots or in fenced plots, based on measurements from fall 2017 in 4 x 1
m belt transects in all plots in the forest. Within the size range, we
selected plants with the least amount of visible insect damage. On 26
July 2018, we identified and marked the plants to be sampled and then
completed the entire foliage collection the next day, on 27 July. We
alternated collection between fenced and unfenced plots, and all
collection was completed for one species before proceeding to the next.
The youngest fully expanded leaf was collected from the plot’s three
plants simultaneously, by having three people work together. The three
leaves were combined, formed into a single pellet, wrapped in foil and
submerged into liquid nitrogen within 30 sec of collection. They were
transferred directly into a -80 C freezer within 2 hr of collection, and
4 days later shipped overnight on dry ice to the Boyce Thompson
Institute for metabolite extraction and analysis.
2.4 |
Extraction and analysis of metabolites
Leaf
samples were ground into fine powder in liquid nitrogen using mortar and
pestle. Two hundred milligrams of the fine powder were transferred into
pre-chilled microcentrifuge tubes that contain two metal beads and
homogenized in 1 mL ice-cold extraction buffer (1: 2: 1 chloroform :
methanol : water; v/v) for 2 min. The homogenized samples were vortexed
for 20 min at 4 oC and centrifuged for 20 min at
15,000 g. Then, 750 µL of the clear supernatants were transferred into
new microcentrifuge tubes and dried under vacuum at room temperature.
After adding 100 µL 70% methanol (in water; v/v), the tubes were
vortexed for 10 min, centrifuged for 10 min at 15,000 g, and 5 µL of the
clean supernatants were analyzed on Q-exactive liquid
chromatography-tandem mass spectrometer (LC-MS/MS; Thermo Scientific) in
negative ionization mode.