Lee Hyoung Yool, Back Kyoungwhan
Department of Molecular Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 61186, Republic of Korea.
Plant J. 2025 Aug;123(3):e70394. doi: 10.1111/tpj.70394.
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.
植物通过酶促反应合成褪黑素及其羟基化形式，如2-羟基褪黑素（2-OHM）和3-羟基褪黑素（3-OHM）。有趣的是，2-OHM和3-OHM在植物中的浓度高于褪黑素，且在夜间被诱导产生，这与动物中褪黑素的夜间诱导情况一致。然而，2-OHM和3-OHM在植物中的生物学功能尚不清楚。在此，我们克隆了拟南芥中负责2-OHM合成的褪黑素2-羟化酶（M2H）基因，并构建了一个缺失M2H和负责3-OHM合成的褪黑素3-羟化酶（M3H）的双敲除突变体（m2hm3h）。M2H和M3H在夜间表达水平最高，但在pif4pif5和bri1中其表达受到极大抑制，而在phyB中它们组成型过表达。因此，M2H和M3H的表达以PIF4/5依赖的方式发挥作用。m2hm3h具有与pif4pif5相似的下胚轴短小表型，并且包括EXP1在内的各种生长相关基因的表达同时降低。PIF4与M2H和M3H启动子的E-box元件结合。此外，与野生型相比，m2hm3h的PIF4转录本和蛋白水平较低，这可能与种子吸胀阶段GA和BR生物合成基因表达的降低有关。为了确定2-OHM/3-OHM对GA和BR生物合成基因的直接影响，种子在黑暗中用2-OHM和3-OHM的混合物进行吸胀处理。与对照相比，种子吸胀期间GA和BR生物合成基因的表达被显著诱导。通过发现PIF4依赖的M2H和M3H表达，我们阐明了2-OHM和3-OHM作为植物暗中形态建成幼苗生长信号分子的作用机制。
