Leaf photosynthesis measurement
Photosynthesis measurements were performed in situ on the fully expanded flag leaf on the main stem of four plants per cultivar in each treatment, 2 weeks after heading. Comparison between the CO2 treatments was made by using an infrared gas analyzers (Li-Cor 6800F; Li-Cor Inc., Lincoln, NE, USA). The leaf photosynthesis rate under saturating light (1,500 μmol m−2s−1) and CO2 levels (1,600 μmol mol−1) was taken using the 6 cm2chamber, as the maximum leaf photosynthesis capacity level (A max). All the measurements were carried out at a leaf temperature of 25 °C, relative humidity in the cuvette set to 65%, with a flow rate of 700 μmol s−1. The steady-state fluorescence yield (Fs) was measured just after registering the gas-exchange parameters, and a saturating light pulse of 8000 μmol m−2 s−1 was applied to achieve the light-adapted maximum fluorescence (Fm’). The operating PSII photochemical efficiency (φPSII) was determined as (1-Fs/Fm’), and then ETR was calculated (Table S1). To minimize the confounding effect of diurnal trends in photosynthesis downregulation related to C sink limitations, previously observed to increase along the day (Fabreet al. , 2019), all measurements were made at least 6 hours after onset of the light period (afternoon). This served to capture effects of photosynthetic acclimation, if any.
Then, the ratio of the average A max of plants grown at e-CO2 by the averageA max of plants grown at ambient CO2 (A max e-CO2 /A max ambient) was calculated.
A relative indicator of chlorophyll content (SPAD) was measured on the same leaf using a SPAD-502 (Minolta, Ltd., Japan). In EXP2, specific leaf area (SLA [cm2 g−1]) was determined in addition, on the flag leaf used for gas exchange measurements. The area of each leaf was measured with a leaf area meter (Li-Cor 3100, Lincoln, NE, USA) and the leaf then oven-dried until constant weight (48 hr at 70°C).