Conductivity meter

Chlorophyll contents in leaves from 2‐wk‐old wild‐type and hts1 mutant plants were measured as previously described (Chen et al., 2018 (link)). Cell death was observed using trypan blue staining as previously described (Wu et al., 2015 (link)). H₂O₂ accumulation was determined by staining with 3,3′‐diaminobenzidine (DAB) (Chen et al., 2010 (link)), or quantified using an Amplex Red hydrogen peroxide/peroxidase assay kit (Invitrogen) following the manufacturer’s instructions. The antioxidant enzyme activities and malondialdehyde (MDA) contents were determined according to Chen et al. (2010 (link)).
Ion leakage was measured as previously described (Shen et al., 2015 ). Briefly, three leaves of 2‐wk‐old rice seedlings harvested after 0, 12, 24 and 48 h of heat stress were trimmed into small pieces and placed in glass tubes containing 10 ml deionized water and incubated overnight at 25°C with shaking (c. 100 rpm). Initial ion leakage (I₀) was determined with a conductivity meter (Mettler Toledo, OH, USA). Total ion leakage (I_t) was measured after 15 min of boiling. Relative ion leakage was expressed as a percentage (I₀/I_t).

In brief, five freshly harvested leaf disks (0.5 cm in diameter) were placed in tubes containing 10 mL of deionized water and incubated at 25 °C on a shaker for 6 h. Then, the initial electrical conductivity was determined using a conductivity meter (Mettler-Toledo Instruments Co., Ltd, Shanghai, China). The final conductivity was measured after boiling at 100 °C for 30 min using previous samples. Relative electrolyte leakage (REL) was determined as the ratio of the initial conductivity to final conductivity [22 (link)]. Chlorophyll pigments were extracted in 80% (v/v) chilled acetone and quantified using a spectrometer (MPDA-1800, Shanghai, China). The absorption spectra of the samples were recorded at 663, 647, and 470 nm wavelength for chlorophyll a, chlorophyll b, and carotenoids, respectively. The absorbance values were converted to concentrations and/or contents according to the experimental equations described by Lichtenthaler [23 ].

Soil physicochemical and enzymatic activity indicators included soil water content (SWC), pH, EC, organic matter content (OM), and total nitrogen content (TN), as well as catalase activity (CAT), sucrose-converting enzyme activity (INV), and urease activity (URE). SWC was calculated after drying at 105°C overnight (Jiao et al., 2023 (link)). The pH was measured with a pH meter (Mettler Toledo) in a 1:2.5 soil:water suspension (Zhang et al., 2013 (link)). The EC was measured with a conductivity meter (Mettler Toledo) in a 1:5 soil:water suspension(Cruz-Paredes et al., 2021 (link)). The OM was determined using the K₂Cr₂O₇ oxidation method (Wang et al., 2022a (link)). The TN was determined using the Kjeldahl method (Guo et al., 2020 (link)). Further, the CAT was determined through titration with KMnO₄ (Wang et al., 2022a (link)). The INV was determined using a 3,5-dinitrosalicylic acid colorimetric assay (Wang et al., 2022a (link)). Finally, the URE was determined via indophenol blue colorimetric method (Yang et al., 2021 (link)).

Leaf disks excised using a cork borer from the fully expanded fourth leaves were utilized for the electrolyte leakage measurements (Lutts et al., 1996 (link)). Seven leaf disks from each treatment were washed with deionized water and then immersed in a test tube containing 10 mL of deionized water for 24 h at room temperature. After that, the initial electrical conductivity (EC1) of the samples was measured using a conductivity meter (Mettler-Toledo GmbH, Greifensee, Switzerland). The same test tubes were then autoclaved (120°C for 20 min) and cooled to room temperature. Then, a second electrical conductivity (EC2) measurement was taken. The EL was calculated using the following formula:

The 20 leaf discs from each treatment were placed in 10 mL of deionized water in a tube. Pump until the blades became transparent and sank underwater. After oscillating the tube at room temperature for 1 h, the initial conductivity value (S1) was measured by conductivity meter (Mettler Toledo, Greifensee, ZÜRICH, Switzerland). Subsequently, they were put in a boiling water bath for 5 min and then were cooled down to room temperature, and the final conductivity was measured (S2).
Relevant calculation formula: $$\mathrm{Relative} \mathrm{conductivity} \left(\mathrm{L}\right)=\frac{{\mathrm{S}}_1}{{\mathrm{S}}_2} \%$$
$$\mathrm{Relative} \mathrm{Electrolyte} \mathrm{leakage} \left(\mathrm{EL}\right) \left(\%\right)=\frac{{\mathrm{L}}_{\mathrm{t}}-{\mathrm{L}}_{\mathrm{CK}}}{1-{\mathrm{L}}_{\mathrm{CK}}}\times 100$$
L_t—Relative conductivity of treated leaves;
L_CK—Relative conductivity of control leaves.

The leaves were histologically stained with DAB for H₂O₂ measurement and trypan blue staining for cell necrosis assays according to a previously published method [32 (link)]. Ionic conductivity was used to quantitatively detect cell death in pepper leaves as described previously [77 (link)]. Six leaf disks with a diameter of 1 cm were taken from the injection area of the leaves and, after soaking in 5 ml ddH₂O for 5 hours, were measured as value A using a conductivity meter (Mettler-Toledo, China). After boiling the centrifuge tube with the leaf disks for 20 minutes, ion conductivity was measured as value B and the ion leakage was calculated as (value A/value B) × 100. H₂O₂ is one of the important reactive oxygen species. The ROS contents of plant leaves were quantified using Micro Hydrogen Peroxide (H₂O₂) Assay Kit (Solarbio, China) with reference to the instructions.