Chilling and MT or NO Treatment

To examine the effect of MT and sodium nitroprusside (SNP, a NO donor) on cold tolerance of cucumber, seedlings with three leaves were foliar sprayed with 0 (control), 25, 50, 75, 100, or 125 μM MT or pretreated with 0 (control), 25, 50, 75, 100, or 125 μM SNP (10 ml per plant). At 24 h after pretreatment with MT or SNP, the seedlings were exposed to 5°C for 72 h for the analysis of malondialdehyde (MDA) content and electrolyte leakage (EL). The seedlings were pretreated with 100 μM MT, 75 μM SNP, or distilled water (control) for examining the effect of MT on endogenous NO generation and that of NO on MT biosynthesis. To analyze the interaction between MT and NO, seedlings with three leaves were pretreated with 100 μM MT, 75 μM SNP, 100 μM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, a specific scavenger of NO) +100 μM MT, 50 μM p-chlorophenylalanine (p-CPA, MT synthesis inhibitor) +75 μM SNP, or deionized water (control). At 24 h after cold stress, the pretreated seedlings were subjected to 5°C in growth chambers. At 24 h after chilling treatment, the gas exchange parameter, fluorescence parameters, and the key enzymes in the Calvin–Benson cycle were detected. At 48 h after cold stress, the accumulation of ROS and gene expression and the activity of antioxidant enzymes were measured. The deionized water treatment under cold stress was considered as H₂O treatment to distinguish from the control at normal temperature conditions.