Decreased anthocyanin accumulation in cyp37-1 mutants under HL conditions. A) Schematic diagram of the CYP37 gene. Purple rectangles indicate exons, black lines indicate introns, and white rectangles indicate untranslated regions (UTRs). Triangles represent T-DNA insertion, and the ATG start codon and TGA stop codon are labeled. Red arrows indicate primers used in RT-PCR. CYP37-F and CYP37-R indicate forward primer and reverse primer used in cyp37-1 genotyping. B) The cyp37-1 mutant was identified by RT-PCR. CYP37 transcripts in the WT and cyp37-1 mutant were reverse transcribed and amplified by PCR. The ACTIN2 gene was used as a reference gene. C) Immunoblotting analysis of CYP37 proteins in WT and cyp37-1 mutant plants. Protein extracts were measured by chlorophyll amount, and the sample containing 3 μg chlorophyll was labeled 1 unit (U). U, 1/2 U, and 1/4 U of protein samples from WT, as well as 1 U protein samples from cyp37-1 mutant, were loaded. Photosystem II subunit O (PsbO) was used as a loading control. D) Anthocyanin accumulation was deficient in cyp37-1 mutant under HL conditions. WT, cyp37-1 mutant, and cyp37-1 RL plants were grown in soil under GL conditions (16 h light/8 h dark, 90 μmol·m⁻²·s⁻¹) for 4 wk (upper panel) and then transferred to HL conditions (10 h light/14 h dark, 350 μmol·m⁻²·s⁻¹) for 7 d (lower panel). The images were digitally extracted for comparison. Scale bars: 1 cm. E) Anthocyanin contents in WT, cyp37-1 mutants, and cyp37-1 RL plants under 350 μmol·m⁻²·s⁻¹ light intensity. Scale bars: 0.5 cm. F) Concentration of anthocyanin in WT, cyp37-1 mutants, and cyp37-1 RL plants under 90 (left panel) and 350 (right panel) μmol·m⁻²·s⁻¹ light intensity. Data represent the means ± Sd, n = 3. **P < 0.01 (Student's t-test). GL conditions and HL conditions were marked with light blue (left panel) and light yellow (right panel) backgrounds, respectively. G) The relative expression of anthocyanin biosynthesis genes in the WT and cyp37-1 mutant under HL (350 μmol·m⁻²·s⁻¹) exposure. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as a reference gene. The distinct anthocyanin biosynthetic pathway in Arabidopsis. Arrows indicate enzymatic reactions; red words indicate anthocyanin biosynthetic genes, and magenta words indicate positive regulators of anthocyanin synthesis. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaryl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid-3′-hydroxylase; DFR, dihydroflavonol reductase; ANS, anthocyanidin synthase; PAP1, production of anthocyanin pigment 1; PAP2, production of anthocyanin pigment 2. Data represent the means ± Sd, n = 3.