Irrigated site: Tthe fertirrigated site (39º2’49.77’’N, 7º57’32.08’’W, UTM coordinates) was installed in 2003 near an intensive olive plantation with some centenary cork oaks scattered throughout the stand, located 35 km from the rainfed site. The fertirrigation system is coupled to the olive plantation. From 2003, the cork oaks were irrigated during the summer, usually for four months. Average watering figures from the cork formation period were 1928.6 m3 ha−1 (2011-2017) and annual precipitation for the same period was 452.19 mm. The plot is located on a Luvisol with 164 trees per hectare, a basal area of 16.93 m2, and an average stem perimeter at breast height of 81.2 ± 40.28 cm. Trees on the plot present a mean ± SD of 8.50 ± 1.76 m of tree height and no decline symptoms. Tree age (years) is 18y + 13 centenary trees.
Rainfed site: A set of permanent plots was installed in 1995 in cork oak forests in the centre of Portugal (39º05’54.93’’N, 8º21’26.23’’W UTM coordinates). Centenary cork oaks were systematically monitored for tree growth and cork production18. The study site presented an annual precipitation of 400.93 mm for the period 2008-2017 and is located on a Cambisol with no limitations on cork oak growth. The site has around of 150 trees per hectare, a basal area of 6.94 m2 and a stem perimeter at breast height (mean ± SD) of 134.6 ± 37.20 cm. The trees showed no decline symptoms and tree height (mean ± SD) is 10.33 ± 2.01 m.
Cork was harvested in 2017 on the two sites. Before harvesting, a 20x20 cm square sample was taken from each truck at a height of 130 cm. The six samples selected for the analysis were from centenary trees: three from each plot. The height (mean ± SD) of the study trees was 11.23 ± 1.68 m for the irrigated plot and 9.93 ± 1.90 m for the rainfed plot. Stem perimeter at breast height (mean ± SD) was 214.3 ± 15.28 cm for the irrigated plot trees and 139.5 ± 45.89 cm for the rainfed plot trees. The harvested cork weight (mean ± SD) was 51.73 ± 7.93 Kg for the irrigated site and 24.9 ± 12.36 Kg for the rainfed site.
Cell Walls - Raw Samples Measurements
Samples prepared for SEM analysis were cut with a movable blade microtome (Reichert, with Jung blades) in the transverse plane (cross section) with 1 mm thickness16. Samples were fixed on aluminium specimen holders using conductive double-sided adhesive carbon tabs and coated with 40nm carbon using the EMITECH K905 Carbon Coater (Emitech Ltd, Ashford, Ken, UK). On each raw sample, one image was gathered using MAPS software (MAPS version 22.214.171.1249, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 10kV beam energy and a spot size of 2.5 under high vacuum conditions using SEM (Quanta FEG 650, Thermo Fisher Scientific, Waltham, Massachusetts, USA). For each sample (6), an image with 168 to 224 subframes was taken with high magnification (Fig. 2). Subframes were stitched together using the MAPS software for image analysis. Cell-wall thickness was measured using ImageJ 1.52a Program (Wayne Rasband, National Institutes of Health, USA). 200 measurements per image were done.
Cell walls - 24h water immersion and 98% humidity measurements
Samples prepared for raw measurement were immersed for 24 hours in cold water and one image was gathered per sample (3 samples per treatment) with MAPS software using ESEM (Quanta FEG 650, Thermo Fisher Scientific, Waltham, Massachusetts, USA) (Fig. 2C an D). Cell-wall swelling was analysed under 98% humidity at 10kV beam energy and a spot size of 2.5 under 98% humidity (800Pa; 4-6°C and working distance of 6,5) within the same area as raw sample measurements. For each sample acquired, 200 measurements were performed using ImageJ 1.52a Program (Wayne Rasband, National Institutes of Health, USA).
Cell walls - after boiling measurements – 1h at 100ºC
Following the cork industrial processing, previous hydrated samples were boiled in water (100 ºC) for one hour and dried under environmental conditions for two days. One image per sample (3 samples per treatment) was gathered using MAPS software in SEM (Fig. 2E and F). 200 measurements were performed using ImageJ, as in previous analyses.
The measurements, in every condition (raw, hydrated and boiled) were done on the same samples and same sample’ spots.
Radial Macro Samples - Boiling Procedure
From the original harvested cork samples, pieces of around 10 cm long and 3 cm thick were cut and scanned using Epson Scan-Expression 11000XL. For each sample, three random lines (Fig. 3) along the radial length were tagged with a permanent marker pen and measured using ImageJ. Samples were boiled at 100o C for one hour, simulating the industrial procedure. After boiling, samples were scanned and radial swelling was measured at the same spots using ImageJ.
All experiments were performed in accordance with relevant named guidelines and regulations. Cork samples and research plots are maintained by University of Évora.