## 2 Materials and Methods  
The study was conducted in the marginal soils (EC = 6.5 ds m\({}^{-1}\), pH = 7.8, CEC = 17 C mol(+) kg\({}^{-1}\)) of the Agricultural and Natural Resources Research and Education Centre of East Azarbaijan (Khosrow-Shah Station), Iran (46\({}^{\circ}\)2\({}^{\prime}\) E, 37\({}^{\circ}\)58\({}^{\prime}\) N), during the growing seasons 2017-2019. The trial was conducted on loamy soil as a split plot based on a randomised complete block design with three replicates. Six winter thistle genotypes were evaluated under non-stressed and under water deficit conditions from mid-flowering to seed maturity. Plants were irrigated at MAD = 35-40% and MAD = 75-80% depletion of available soil water in the non-stressed and stressed plots, respectively (Table 1). The EC value of the irrigation water was 2.7 ds m\({}^{-1}\). The prevailing weather conditions during the growing seasons are summarised in Table 2. The plot size was 5 \(\times\) 1.8 m. The distance between the rows was 30 cm and the plants were placed 10 cm apart in a row. The sowing date was 15 September for the two years of the experiment. The youngest, fully unfolded leaves were used to determine the physiological parameters. To calculate the RWC, the detached leaf discs (3 discs with a diameter of 20 mm) were first weighed as fresh weight (FW) and then the threshold weight (TW) was determined by floating them in distilled water at 5 \({}^{\circ}\)C for 4 h under low light. The dry weight (DW) was determined after the samples had been dried at 80 \({}^{\circ}\)C for 24 h. Fresh weight (FW), TW and DW were used to calculate RWC as RWC = FW - DW/TW - DW [26; 27]. Stomatal conductance (gs) was determined using an AP4 leaf porometer (Delta-T Devices, Cambridge, UK). An infrared thermometer (class 2, Testo, Kirchzarten, Germany) was used to measure crown temperature. Its emissivity was set to 0.56 for the green leaf surface. Measurements were taken on five samples from each plot on the youngest fully developed leaves at 1200 to 1400 h [28; 29]. The Crop Water Stress Index (CWSI) was determined using the following equation: CWSI = Tc \(-\) Twet/Tdry \(-\) Twet, where Tc is the actual canopy temperature, Twet (fully transpiring leaf with open stomata) is the temperature of leaves in the canopy that were sprayed with water on both sides one minute before measurement, and Tdry (non-transpiring leaf with closed stomata) is the temperature of leaves in the canopy that were laterally shielded with Vaseline oil [30; 31]. The water potential (WP) of the leaves was measured using a DIK-7000 pressurised chamber (Ogawa Seiki Co., Ltd., Tokyo, Japan). Osmotic adjustment (Oadj) was determined according to the method of [PERSON] [32]. Some of the leaf samples used to determine RWC were separated and placed in liquid nitrogen for rapid freezing. After thawing, the samples were stored at \(-\)18 \({}^{\circ}\)C, the juice was extracted with a press and the osmotic potential (OP) of the sample was measured with an osmometric vapour pressure chamber (Westcor Inc., Logan, UT, USA) connected to a microvoltmeter. \begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & **FC (\%)** & \multicolumn{2}{c}{**PWP (\%)**} & \multicolumn{2}{c}{**AWC (\%)**} \\ \hline
**Soil Depth (cm)** & **Year** & **2017–2018** & **2018–2019** & **2017–2018** & **2018–2019** & **2017–2018** & **2018–2019** \\ \hline
0–30 & 26.7 & 25.2 & 12.6 & 12.8 & 14.1 & 12.4 \\
30–60 & 20.8 & 20.4 & 10.0 & 11.4 & 10.8 & 9.0 \\
60–90 & 13.7 & 13.1 & 7.5 & 8.0 & 6.2 & 5.1 \\ \hline \hline \end{tabular}  
* FC = field capacity, PWP = permanent wilting point, AWC = available water capacity.  
\end{table} 
Table 1: Characteristics of soil water content in the experimental field (Khosrow-Shah Research Station, Iran). The OP at full turgor (OP100) was then estimated from the measured OP and the RWC by OP100 = OP \(\times\) (RWC/100).