Lack of CYP37 leads to decreased chlorophyll content and increased ROS accumulation under HL and oxidative stress. A) Chlorophyll degradation in cyp37-1 mutant plants under HL stress. Wild type (WT), cyp37-1, and RL plants grew on 1/2 MS medium in growth chambers (16 h light/8 h dark, 90 μmol·m⁻²·s⁻¹) for 2 wk, following GL conditions (16 h light/8 h dark, 90 μmol·m⁻²·s⁻¹) (left panel), as well as HL stress (10 h light/14 h dark, 350 μmol·m⁻²·s⁻¹) (middle panel) and HL stress (10 h light/14 h dark, 750 μmol·m⁻²·s⁻¹) (right panel) for 2 d. B) Chlorophyll concentrations in the WT, cyp37-1, and RL plants under GL (90 μmol·m⁻²·s⁻¹) and HL conditions (350 μmol·m⁻²·s⁻¹) for 2 d. FW, fresh weight. Data represent the means ± Sd, n = 3. *P < 0.05 (Student's t-test). C) Detection of oxidative stress by MV treatment showed decreased chlorophyll caused by accumulated ROS in cyp37-1. Rosette leaves of 3-wk-old WT, cyp37-1, and RL seedlings supplemented with 0, 3, and 5 μM MV for 2 d. D) Total chlorophyll content of the recovered seedlings after prolonged exposure to 1/2 MS medium containing the indicated content of MV. WT, cyp37-1, and RL seedlings growing on ½ MS medium for 10 d were transferred to ½ MS medium containing the indicated concentration of MV for another 2 d in growth chambers (16 h light/8 h dark, 90 μmol·m⁻²·s⁻¹). Data represent the means ± Sd, n = 3. *P < 0.05 (Student's t-test). E) Mutant cyp37-1 accumulated more ROS in cells under HL intensity. Intracellular ROS accumulation in WT and cyp37-1 mesophyll protoplasts under HL treatment for 15 min. Protoplasts extracted from WT and cyp37-1 plants were subsequently treated with or without HL (2000 μmol·m⁻²·s⁻¹) and incubated with the ROS-sensitive fluorescent dye 2′, 7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) at a final concentration of 5 µM for 5 min at room temperature. Fluorescence signals were determined by confocal microscopy as described in the Materials and Methods. DCF (the oxidation product of H₂DCFDA) fluorescence was falsely colored green. BF, bright-field image under transmitted light; Merge, merged image of DCF and bright field. Scale bars: 10 μm. F) ROS accumulation in cyp37-1 mutant plants under HL (750 μmol·m⁻²·s⁻¹) stress. Two-wk-old WT and cyp37-1 mutant plants were subjected to HL treatment for 5 h. NBT staining was used to assess the superoxide anion. DAB staining was used to determine the hydrogen peroxide content. G) Mutant cyp37-1 accumulated more ROS in leaves under oxidative stress. Representative images of H₂O₂ accumulation as determined by NBT and DAB staining after MV treatment. H) The relative expression levels of the ROS marker genes FeSODs and APXs were measured by RT-qPCR in WT and cyp37-1 after HL (350 μmol·m⁻²·s⁻¹) treatment. The results were normalized to GAPDH. FSD, Fe-dependent superoxide dismutases, catalyzing O₂^.− to H₂O₂; APX, ascorbate peroxidase, catalyzing the reduction of H₂O₂ into water. APX1&APX2, cytosolic APX; tAPX, thylakoid membrane-bound APX; sAPX, stromal APX. Data represent the means ± Sd, n = 3.