Experimental Methods: This experiment occurred in Fort Collins, CO at the USDA-ARS Crops Research Laboratory greenhouse complex. We used two greenhouse bays and set the CO2 concentrations for each bay to either 400 ppm or 800 ppm. The bays were maintained at a temperature of 30°C during the day and 21°C at night with a 12-hour photoperiod. The bays were lit with HID lighting. We grew four different woody plant species (Cornus drummondii, Gleditsia triacanthos, Juniperus osteosperma, Rhus glabra) in a blocked design with CO2 concentrations treatments in the two separate bays, and nested within each bay was a chronic water stress treatment and control water treatment. The water treatments were randomized within each bay. We also rotated the CO2 treatments and the plants subject to those treatments between the bays biweekly. Native soils were used for each woody plant. The water treatments were determined based on the volumetric water content from each pot and species throughout the study. The chronic water stressed plants never had a volumetric water content more than 20% for C. drummondii, G. triacanthos, R. glabra and 10% for J. osteosperma because of soil texture. The control plants were always watered to fully saturated soils. Watering occurred once a week.
Data Collection Methods:
1). Gas Exchange: Instantaneous gas exchange rates were taken using a Li-6400XT open system gas analyzer (Li-Cor, Inc., Lincoln, NE). Gas exchange measurements were taken every two weeks from 1000-hrs to 1600 hrs for the full duration of the experiment. Leaves that did not fill the cuvette chamber were photographed on 1 cm2 grid paper sing an iPhone 5c camera (8 MP) and then leaf area estimated using imageJ software to correct the gas exchange parameters measured. CO2 concentrations were matched to the greenhouse bay to match the CO2 treatment (400 ppm, 800 ppm).
2). Biomass: At the end of the greenhouse experiment all woody plant species were cleaned with DI water to remove particulates, separated by tissue type (i.e., roots, stems, leaves) and dried for 72 hours at 60oC before being weighed.
3). Starch Analysis: Several grams of tissue were harvested from each woody plant species at the end of the greenhouse experiment. All tissues were cleaned in DI water to remove all dirt impurities, microwaved for 90 seconds, and then dried for 48 hours at 60oC before being ground using a Wig-L-Bug® grinder. Then all of the samples had the starch extracted and were analyzed colormetrically using a spectrophotometer (O’Connor et al. 2020. Ecology 101:e02935).
