3 cm2 leaf chamber

The measurements of Pn and E were carried out after 3, 6, and 9 days of treatments from approximately 5 to 9 h following the onset of photoperiod and by alternating plants from the different treatments. Three fully expanded uppermost leaves from each plant were marked and used for the measurements using a LI-6400 portable photosynthesis system with a 2 × 3 cm² leaf chamber (LI-COR Biosciences, Lincoln, NB, USA). The reference CO₂ concentration was 400 μmol mol⁻¹, the flow rate was 200 μmol s⁻¹, and the relative humidity (RH) level was set to 50% in the cuvette. The leaf chamber temperature was maintained at 20 °C, and PPFD was 400 μmol m⁻² s⁻¹ provided by the red-blue light spectrum of the light attachment. To determine leaf areas, the parts of the leaves that were inserted into the leaf chamber were excised after the last measurement and scanned. The leaf areas were calculated using the Sigmascan Pro 5.0 computer software (Systat Software, San Jose, CA, USA).

After three days of hypoxia treatment, net photosynthesis rate (P_n), stomatal conductance (g_s), and transpiration rate (E) were measured from three to six hours following the onset of the photoperiod using a Licor-6400 portable photosynthesis system with a 2  ×  3 cm² leaf chamber (LI-COR, Lincoln, NB, USA). The reference CO₂ concentration was set to 400 μmol mol⁻¹, and the flow rate was 200 μmol s⁻¹. The leaf chamber temperature was maintained at 20 °C, and the PPFD was 400 μmol m⁻² s⁻¹ of the red-blue light spectrum. Six to eight plants from each group were randomly selected and three fully expanded uppermost leaves from each plant were measured. The average values of the three leaves were used to calculate the means. After the measurements, the leaves were excised and immediately placed into a Scholander pressure chamber (PMS Instruments, Corvallis, OR, USA) for the measurements of leaf water potential (Ψ_leaf) [61 (link),62 (link)] (n = 6). Dry weights of plants were determined after one, three, eight, and twelve days of hypoxia in plant tissues dried in an oven at 80 °C for 72 h (n = 6).