Leaf-level gas-exchange responses measured with the LI-6800 system on chlorina and TM2016 mutants

Analyses were performed on plants grown at the University of Tartu. Seeds were placed between wet tissue papers and germinated at room temperature, in darkness, for 2-3 days, and transferred on peat:vermiculite:water (4:2:3) growth substrate. Plants were grown at 530 μmol photons m−2 s−1 (PPFD) for 16 h light/8 h dark. Day/night temperatures were set to 20/18 °C, with a relative humidity of 60%. Plants were watered uniformly throughout the experiment and analysed at 16 DAS. Light-response and CO2-response (A/Ci) curves were determined using a LI-6800 portable photosynthesis system (LI-COR Biosciences). Fully expanded leaves were enclosed in a 1 × 3 cm leaf chamber and dark-adapted for 20 min prior to measurements. Chamber conditions were maintained at 23 ◦C leaf temperature, 60% relative humidity, and an ambient CO2 concentration of 420 ppm. Illumination was provided by the integrated LED light source using a red/blue ratio of 90:10. For light-response curves, leaves were exposed to increasing irradiance levels (0, 50, 150, 300, 600, 900, 1200, and 1500 μmol photons m−2 s−1; PPFD). At each light intensity, leaves were allowed to equilibrate for 10–15 min before net CO2 assimilation rate and stomatal conductance were recorded. The saturating irradiance determined from the light-response curves was subsequently used for CO2-response measurements. After stabilization of photosynthetic activity, leaves were exposed to a sequence of increasing CO2 concentrations (400, 300, 200, 100, 30, 400, 600, 800, 1000, and 1200 ppm; Ci), allowing 45–90 s of equilibration at each step before recording gas-exchange parameters. Net CO2 assimilation rate and stomatal conductance were used to assess the response of photosynthesis to changes in CO2 availability.