Stress Tolerance Assays for Nicotiana nudicaulis

Four-week-old seedlings of N. nudicaulia were used for assays of heat stress tolerance. Seedlings of N. nudicaulia were cultured in a growth chamber under a 14 h light/10 h dark photoperiod at 23 °C. For the heat stress treatment, the temperature of the light incubator was adjusted to 48 °C for 6 h. Electrolyte leakage (EL) of leaves was measured according to Sun et al. (2018) [59 (link)]. According to previous method, tobacco leaves were excised and scored according to their fresh weight, rehydrated and dry weights, and RWC = (fresh weight − dry weight)/(rehydrated weight−dry weight)”, and this was used to calculate RWC of leaves [60 (link)]. Chlorophyll content was determined by UV-2250 spectrophotometer (Shimadzu, Kyoto, Japan) after leaf chlorophyll was extracted with 80% acetone, as described by Liu et al. (2021) [61 (link)]. The activities of SOD, POD, and the MDA, H₂O₂, and O²⁻ content, were detected with their corresponding detection kits (Suzhou Comin Biotechnology Co., Ltd., Suzhou, China). Each experiment was repeated three times.