HighlightTransgenic rice plants either overexpressing or suppressing rice endogenous caffeic acid O-methyltransferase (COMT) resulted in an increase or a decrease, respectively, in melatonin levels, suggesting a direct role of COMT on in vivo melatonin synthesis.

In plants, melatonin production is induced by stimuli such as cold and drought, and cadmium (Cd) is the best elicitor of melatonin production in rice. However, the mechanism by which Cd induces melatonin synthesis in plants remains unknown. We challenged rice seedlings with Cd under different light conditions and found that continuous light produced the highest levels of melatonin, while continuous dark failed to induce melatonin production. Transcriptional and translational induction of tryptophan decarboxylase contributed to the light induction of melatonin during Cd treatment, whereas the protein level of light-induced caffeic acid O-methyltransferase (COMT) was decreased by Cd treatment. In analogy, COMT enzyme activity was inhibited in vitro by Cd in a dose-dependent manner. Notably, the Cd-induced melatonin synthesis was significantly impaired by treatment with either an H O production inhibitor (DPI) or an NO scavenger (cPTIO). The combination of both inhibitors almost completely abolished Cd-induced melatonin synthesis, suggesting an absolute requirement for H O and NO. However, neither serotonin nor N-acetylserotonin (NAS) was induced by H O alone. In contrast, NO significantly induced serotonin production but not NAS or melatonin production. This indicated that serotonin did not enter chloroplasts, where serotonin N-acetyltransferase (SNAT) is constitutively expressed. This suggests that chloroplastidic SNAT expression prevents increased melatonin production after exposure to stress, ultimately leading to the maintenance of a steady-state melatonin level inside cells.

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants.

Cyclic 3-hydroxymelatonin (c3OHM) is a major metabolite of melatonin in plants produced by the enzymatic action of melatonin 3-hydroxylase (M3H). However, the function of c3OHM in plants is unclear. Here, we report that M3H mRNA and c3OHM levels display diurnal rhythms with peaks at night, but not in a circadian manner. This diurnal rhythmicity occurred predominantly in the late vegetative growth stage (8 weeks after germination), but was absent in the early vegetative growth stage. Transgenic rice plants overexpressing or underexpressing M3H were generated to investigate the physiological roles of diurnal production of c3OHM. The M3H-overexpression (OE) line exhibited higher M3H activity and c3OHM production than the wild-type, and vice versa for the M3H‑underexpression rice (RNAi). The seedling growth phenotype of the OE and RNAi lines was comparable to that of the wild-type but exhibited pleiotropic phenotypic defects at the reproductive stage, such as decreased height, biomass, grain yield, and fertility. Of note, the OE rice showed significantly increased numbers of secondary tillers and panicles. The increase in tiller number of the OE line was linked to increased expression of tiller-related genes, such as MOC1 and TB1, suggesting that the diurnal rhythm of c3OHM production is associated with the tiller number, a pivotal agronomic trait governing grain yield in rice.

Melatonin 2-hydroxylase (M2H) catalyzes the conversion of melatonin into 2-hydroxymelatonin (2OHM), which is present in plants at a higher concentration than melatonin. Although M2H has been cloned, the in vivo function of its product is unknown. Here, we generated stable T2 homozygous transgenic rice plants in which expression of endogenous M2H was suppressed (RNAi lines). However, we failed to generate M2H overexpression transgenic rice due to failure of somatic embryogenesis. The M2H transcript level showed a diurnal rhythm with a peak at night concomitantly with the peak concentration of 2OHM. RNAi rice showed a reduced M2H mRNA level and 2OHM and melatonin concentrations. The unexpected decrease in the melatonin concentration was caused by redirection of melatonin into cyclic 3-hydroxymelatonin via a detour catabolic pathway. Thus, the decrease in the melatonin concentration in M2H RNAi rice led to slowed seedling growth and delayed germination. By contrast, the transient increase in the melatonin concentration was of greater magnitude in the M2H RNAi than the wild-type rice upon cadmium treatment due to possible suppression of melatonin degradation. Due to its higher concentration of melatonin, the M2H RNAi rice displayed tolerance to senescence, salt, and tunicamycin stresses. Therefore, the increase in the melatonin concentration caused by suppression of melatonin degradation or by overexpression of melatonin biosynthetic genes enhances stress tolerance in rice.