Leaf Alcohol Insoluble Residue (AIR) preparations
Cell wall preparations (alcohol insoluble residue; AIR), were created from poplar leaf samples collected during the drought and13C2-acetate labeling experiments. Leaves were flash frozen in liquid nitrogen and then ground to a powder with a pestle and mortar on dry ice. The ground samples were incubated in 96% (v/v) ethanol at 70°C for 30 minutes. The supernatant was discarded and the samples washed successively in 100% ethanol, 2:3 chloroform: methanol (twice, with shaking for at least 1 hour), 60% ethanol, 80% ethanol and 100% ethanol. Samples were centrifuged and the supernatant discarded between each washing step. The resulting AIR was dried in a speedvac and destarched for the monosaccharide analyses using amylase, amyloglucosidase and pullunanase (Megazyme Ltd., Ireland) as previously described (Sechetet al., 2018).
Bulk O -acetyl ester content of AIR samples was carried out using a commercial kit (Acetate Assay Kit, BioVision, CA, USA). AIR samples (2.5 mg) were saponified with NaOH (1 M, 125 µL) for 16 hours then neutralized with 1 M HCl. The samples were centrifuged (10 minutes at 15000 rpm) and 5 µL of the supernatant was transferred to a 96-well plate. The samples were treated with the assay kit enzymes and plates incubated at room temperature for 40 mins. Absorbances were measured at 450 nm on a 96-well plate reader (SpectraMax M2; Molecular Devices, CA, USA). Total O -acetyl content of the AIR samples (µg/mg AIR) were determined by including a six-point calibration on each plate using the included standard.
In order to determine bulk leaf cell wall monosaccharide composition, destarched AIR (200 µg) was incubated in 2M trifluoroacetic acid (400 µl) at 120°C for 3 hours. The supernatant was collected after centrifugation. The pellet was washed with 200 µl milliQ water, centrifuged and the supernatant collected. The combined supernatants from each sample were dried in a speedvac. The sample was resuspended in 200 µl milliQ water, filtered on a 0.22 µm centrifuge filtration plate then analyzed for monosaccharide composition using high-pressure anion-exchange chromatography (Dionex-ICS 5000, Dionex, CA, USA).