Centrifuge 5425 5425 r

A 0.5 g of leaves were homogenized in 6 mL of phosphate buffer (65 mM, pH 7.8) and then centrifuged at 10,000 rpm (Centrifuge 5425/5425 R; Eppendorf, Germany) at 4 °C for 10 min. Next, 1 mL of supernatant, 0.9 mL of phosphate buffer (65 mM, pH 7.8), and 0.1 mL of hydroxylamine hydrochloride (10 mM) were mixed, and the solutions were placed in a water bath for 20 min at 25 °C. To 0.5 mL of those solutions, 0.5 mL of sulfanilic acid (17 mM) and 0.5 mL of α-naphthylamine (17 mM) were added, followed by 20 min of incubation at 25 °C in a water bath. O₂^•− production rate was determined following the addition of N-butyl alcohol and read at 530 nm, which was expressed as μM min⁻¹ g⁻¹ FW [54 (link)]. A 0.5 g of leaves was homogenized in 5 mL of ice-cold trichloroacetic acid (0.1%, w/v) and centrifuged at 12,000 rpm (Centrifuge 5425/5425 R; Eppendorf, Germany) at 4 °C for 15 min to collect the supernatant, then optical absorption of the supernatant was measured spectrophotometrically at 410 nm to measure H₂O₂ content and expressed as μM g⁻¹ FW [55 (link)].

A 0.1 g of leaves were digested in 5 mL alcohol (95%, v/v) overnight at 4 °C, and then centrifuged at 12,000 rpm (Centrifuge 5425/5425 R; Eppendorf, Germany) for 5 min at 4 °C. The supernatant was stored at 4 °C for Chl a, Chl b, Chl a+b, and Chl a/b ratio measurements [46 (link)]. Leaf photosynthetic parameters were measured between 10:30 and 11:30 AM using an LI-6400 XT portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). The Pn, Ci, Gs, and Tr were measured in a chamber at 1,500 μM m⁻² s⁻¹ photosynthetically active radiation (PAR) and 380 ± 5 μM CO₂ M⁻¹ [15 ]. The WUE and Rubisco activity [47 (link)] were estimated as follows:

A 0.1 g of leaves was placed in test tubes containing 10 mL of double–distilled water and incubated at 40 °C for 30 min. The conductivity (C₁) was measured using a DDSJ-308F conductivity meter (Rex Electric Chemical, China). The same set was then kept in a water bath at 100 °C for 15 min, and the conductivity was recorded (C₂). The MSI [20 (link)] was calculated as follows:
The lipid peroxidation was measured by MDA content [53 (link)]. Briefly, a 0.5 g of leaves was homogenized in 5 mL of 0.3% thiobarbituric acid (TBA) and 10% (v/v) trichloroacetic acid. After incubation at 100 °C for 30 min, mixtures were centrifuged at 12,000 rpm (Centrifuge 5425/5425 R; Eppendorf, Germany) for 10 min. The absorbance of the colored supernatant was measured at 450 nm, 532 nm, and 600 nm, respectively. Then MDA concentration was calculated and its content was expressed as µM g⁻¹ fresh weight (FW).