2.2 | A-Ci curve measurements
Between 31 August and 14 September 2017 we measured A-Cicurves on sun-exposed leaves of T. rosea saplings over a wide, but realistic leaf temperature range (i.e., no temperatures lower than minimum daytime temperatures were included to avoid cold stress responses in these lowland tropical plants). In the control dome the measurement leaf temperature ranged from 26.5 to 43.3°C; in the treatment dome the range was 28.0–44.6°C. Target temperatures varied based on the leaf temperatures reached and the requirements to fill the gaps in the curves. Dome temperatures were maintained as close as possible to target leaf temperatures to minimize the temperature differential between the measured leaf and the rest of the plant. The block temperature of the leaf cuvette was controlled using the Peltier cooling/heating capacity of the LI-6400XT portable photosynthesis system (LI-COR Biosciences, Lincoln, Nebraska, USA). Leaf temperature always exceeded air temperature because of the heat generated by the light source; hence the lowest measurement temperature was higher than the lowest ambient daytime air temperature.
Fully-expanded mature sun-exposed leaves that had developed under the growth conditions in the dome were selected from the second-or third most recently emerged leaf pair and each leaf was measured 2–18 times (average 12, median 14). Measurements were made on leaves between 0.5 and 1.0 m above ground level. A-Ci curves were measured at a pre-determined light saturation level of 1500 µmol m–2s–1 that was provided by the red:blue LED light source of the LI-6400XT. Light-saturated photosynthesis rates were determined at ≥13 CO2 concentrations between 50 and 1950 ppm, using the built-in CO2 mixer of the LI-6400XT to control CO2 concentrations of the incoming air, which was delivered at a flow rate of 500 µmol s–1. Leaf temperature during measurements was monitored abaxially with a Type E thermocouple inside the leaf cuvette. After equilibration at ambient [CO2] in the dome, photosynthesis was recorded at 50, 100, 200, 300, 400, 475, 550, 675, 800, 950, 1200, 1500 and 1950 ppm CO2 of the incoming air. Measurements were discontinued whenever a precipitous decline in stomatal conductance was observed and photosynthesis rates failed to stabilize at a given target CO2 concentration.
After completing these measurements of plants at their respective growth conditions, control plants were moved to the treatment dome, and treatment plants to the control dome. At this point 46 useable A-Ci curves had been collected for control plants, and 46 curves for treatment plants. After one week at their new condition, we started re-measuring the same plants (and the same leaves). The post-transition measurements yielded another 46 A-Cicurves for control plants transferred to treatment conditions and 44 curves for treatment plants transferred to control conditions.
Before calculating VCMax and JMax, we corrected photosynthesis rates and Ci values for diffusion errors associated with CO2 concentration gradients between the leaf cuvette and the atmosphere. To do so, we measured CO2-response curves in both domes for leaves that were killed by submersion in boiling water for ~5 minutes. These measurements were taken at 30°C, as previous tests showed no temperature effect on the diffusion error (Slot & Winter 2017b). Applying the correction changed VCMax and JMax estimates by 5 and 7% (median), respectively, but had no effect on the temperature responses of these parameters.