Drought Stress Experiment

Drought stress was applied as described earlier (Daszkowska-Golec et al., 2017). Briefly, the experiment was carried in boxes (400 x 140 x 175 mm) filled with soil containing a mixture of sandy loam and sand (7:2) with known physicochemical properties. The soil was supplied with nutrient medium (Supplementary Material S1). Based on performed calculations, the water was easily available for plants at 14% of volumetric water content (vwc) in the soil, whereas the severe drought stress was achieved at 1.5%. When plants were grown in 14% vwc, the RWC in leaves was approximately 100%. The soil moisture was measured every day using time-domain reflectometer (TDR) EasyTest (Institute of Agrophysics, Polish Academy of Sciences, Poland). The WT and mutant plants were grown in a greenhouse for 10 d after sowing (DAS) under optimal water conditions (14% vwc), 20/18°C day/night, with a 16/8 h photoperiod, and 420 μE m⁻² s⁻¹ light intensity which was provided by fluorescent lamps. Afterward, the soil moisture was decreased by withholding the irrigation under the control of TDR measurements. On 15 DAS, when the soil moisture decreased to 3%, the plants were moved into a growth chamber, where temperature regime was set to 25°C/20°C day/night, with a 16/8 h photoperiod and 420 μEm⁻² s⁻¹ light intensity. The severe drought stress (1.5% vwc) lasted 10 d (16–25 DAS). The control plants were grown under the same conditions with optimal water supply (14% vwc) in parallel to the drought treated plants. On 25 DAS the RWC was evaluated for each genotype, according to the procedure described below.

The second leaves were collected from hvabi5.d and WT plants before water withdrawal (10 DAS) and after severe drought (25 DAS) and used for RNA extraction. The anthocyanins content index, flavonols content index, chlorophyll a fluorescence, and chlorophyll content index were analyzed before water withdrawal (10 DAS) and after drought stress (25 DAS). The stomatal conductance was measured under optimal water conditions (10 DAS), on the drought onset (13 and 15 DAS) and after severe drought (25 DAS). On the 25 DAS, RWC, electrolyte leakage (EL), and ABA content were analyzed using plants growing under optimal water supply and under drought stress. The schedule of drought experiment with indicated time-points of all assays are presented in Supplementary Material S2. We conducted all analyses using the second leaf since it was already present when plants entered drought treatment. Each genotype was tested in three biological replicates. One box containing 15 plants per genotype was considered as one replicate.

RWC was measured in the second leaf on the last day of drought stress (25 DAS). RWC parameter was calculated according to formula:

where: F_w (fresh weight) is the weight of detached leaf, T_w (turgid weight) is the weight of a leaf after 24 h rehydration in distilled water (leaves were submerged in distilled water in darkness), and D_w (dry weight) is the weight of a leaf dried at 60°C for 48 h. The measurement of RWC was performed in three biological replications (each biological replicate included leaves of three independent seedlings).

The EL was analyzed in drought-treated and control seedlings on 25 DAS (according to the protocol of Bandurska and Gniazdowska-Skoczek, 1995). Briefly, the fragments of the middle part of the second leaf were washed three times in deionized water and kept at 10°C for 24 h. Afterward, the samples were transferred to the room temperature and EL was measured using pH/conductivity meter (CPC-505, Elmetron, Poland). Then, samples were autoclaved for 15 min to damage cells completely and conductivity was measured again at room temperature. Three replications were made for each treatment combination (each biological replicate included leaves of three seedlings). EL was calculated according to formula:

Where: D1 and C1 are the first conductivity measurements for respectively drought and control samples, whereas D2 and C2 are the second (after auctoclaving) conductivity measurements.

The measurements of flavonol, anthocyanin, and chlorophyll content indices were performed on the second leave on the 10 and 25 DAS using a Dualex Scientific+TM (Force-A, France). The measurements were performed in three biological replicates, with four seedlings per replicate.

Stomatal Conductance (mmol m⁻² s⁻¹) was measured using AP4 porometer (DELTA-T Devices, Burwell, UK). Measurements were conducted in three biological replicates, each replicate contained three individual plants. The analysis was performed at the exposed, central part of the second leaf adaxial side on 10, 13, 15, and 25 DAS, in three biological replications (each biological replicate included leaves of three independent seedlings).

ABA content was measured according to the protocol of Nakurte et al. (2012). A modular HPLC system (Shimadzu, Japan) with SPD-M20A photodiode array detector and a Kinetex™ C18 (4.6 × 250 mm, 5 µm) column (Phenomenex) were used to conduct chromatography. The injection volume was 20 µl and the analysis was performed in the isocratic mode at a flow rate of 1 ml min⁻¹. LabSolutions software was used to evaluate the results (Shimadzu, Japan).