Morpho-physiological changes imposed by water deficit in cork oak saplings

Cork oak half-sibling acorns from two field-grown mother trees (Alcochete, Portugal, Lat. 38.747491; Long. -8.931877) were collected in November 2018. Viable acorns were selected and cleaned by submergence in tap water, transferred to a water bath at 45 ºC for 2 h and dried in a 30 ºC chamber for 24 h. Acorns were stored in plastic bags at 4 ºC in the dark until further use. In January 2019, acorns were sown on a mixture of sand and vermiculite (1:1) in 700 mL pots (1 per pot) and maintained under a 12 h photoperiod (light intensity 400 μmol m-2 s-1) at 25 ºC, with regular watering. After two months, saplings were transferred to 7 L pots containing a mixture of soil and sand (1:1) and grown from March 2019 to September 2020 (assay-1) in a greenhouse (Oeiras, Portugal, Lat. 38.696189, Long. -9.320762). During the first year of growth all plants were kept under well-watered (WW) conditions (300 mL H2O/day) for optimal growth. In March 2020, saplings were divided into two groups that were subjected to WW conditions (control) or water deficit (WD), imposed by 4 weeks of water withholding followed by 20 weeks of deficit irrigation (300 mL H2O /week). A total of 57 plants were included in each group. Soil water content (SWC) was followed during the experiment and calculated according to (Sapeta et al., 2013). After stress imposition, stem diameter (1 cm above the root collar) and main stem height were measured monthly. A replicate assay (assay-2) was conducted with cork oak saplings sown in January 2021. In this case, drought stress was applied as mentioned above, from March 2022 to August 2022. These plants were used to measure photosynthesis efficiency using chlorophyll fluorescence. The effect of drought on photosynthesis was evaluated in assay-2 by rapid photosynthetic light curves using a MINI-PAM-II fluorometer (Heinz-Walz, Germany). This analysis was conducted in stems and leaves at day 0, 56, 100 and 120 after stress imposition. In detail, chlorophyll a fluorescence was measured in dark-adapted young leaves and stem sections (1 cm above root collar) subjected to increasing (every 20 seconds) photosynthetic active radiation (PAR, 3 – 470 μmol photons m-2 s-1). The efficiency of Photosystem (PS) II (ΦPSII) was calculated using the formula ΦPSII = (Fm’-Fs)/Fm’ (Genty et al., 1989), where Fs and Fm’ represent steady state and maximum fluorescence, respectively. Electron transport rate (ETR) was calculated using ETR = ΦPSII x PAR x 0.5 x 0.84, where 0.84 is the assumed light absorbance of the leaf, and 0.5 the fraction of light absorbed by PSII (Genty et al., 1989).