PIF4-dependent 2-hydroxymelatonin and 3-hydroxymelatonin biosynthesis is involved in skotomorphogenic seedling growth in Arabidopsis thaliana.

Plants enzymatically synthesize melatonin and its hydroxylated forms, such as 2-hydroxymelatonin (2-OHM) and 3-hydroxymelatonin (3-OHM). Interestingly, 2-OHM and 3-OHM are present in plants at higher concentrations than melatonin and are induced at night, which is consistent with the nocturnal induction of melatonin in animals. However, the biological functions of 2-OHM and 3-OHM in plants are unclear. Here, we cloned the melatonin 2-hydroxylase (M2H) gene responsible for 2-OHM synthesis in Arabidopsis and generated a double knockout mutant (m2hm3h) lacking M2H and melatonin 3-hydroxylase (M3H), which is responsible for 3-OHM synthesis. M2H and M3H were expressed at maximum levels at night, but their expression was greatly suppressed in the pif4pif5 and bri1, whereas they were constitutively overexpressed in phyB. Therefore, M2H and M3H expression act in a PIF4/5-dependent manner. m2hm3h had a short hypocotyl phenotype similar to the pif4pif5 and showed simultaneous decreases in the expression of various growth-related genes, including EXP1. PIF4 bound to the E-box element of the M2H and M3H promoters. In addition, m2hm3h showed lower PIF4 transcript and protein levels compared to the wild type, which may be related to the reduced expression of GA- and BR-biosynthetic genes at the seed imbibition stage. To determine the direct effects of 2-OHM/3-OHM on GA and BR biosynthesis genes, seeds were imbibed with a mixture of 2-OHM and 3-OHM in darkness. The expression of GA and BR biosynthesis genes was significantly induced during the seed imbibition period compared to the control. By discovering PIF4-dependent M2H and M3H expression, we elucidate the mechanism by which 2-OHM and 3-OHM act as signaling molecules for skotomorphogenic seedling growth in plants.