We investigated the expression patterns of genes involved in melatonin synthesis and degradation in rice leaves upon cadmium (Cd) treatment and the subcellular localization sites of melatonin 2-hydroxylase (M2H) proteins. The Cd-induced synthesis of melatonin coincided with the increased expression of melatonin biosynthetic genes including tryptophan decarboxylase (TDC), tryptamine 5-hydroxylase (T5H), and N-acetylserotonin methyltransferase (ASMT). However, the expression of serotonin N-acetyltransferase (SNAT), the penultimate gene in melatonin biosynthesis, was downregulated, suggesting that melatonin synthesis was counter-regulated by SNAT. Notably, the induction of melatonin biosynthetic gene expression was coupled with the induction of four M2H genes involved in melatonin degradation, which suggests that genes for melatonin synthesis and degradation are coordinately regulated. The induced M2H gene expression was correlated with enhanced M2H enzyme activity. Three of the M2H proteins were localized to the cytoplasm and one M2H protein was localized to chloroplasts, indicating that melatonin degradation occurs both in the cytoplasm and in chloroplasts. The biological activity of 2-hydroxymelatonin in the induction of the plant defense gene expression was 50% less than that of melatonin, which indicates that 2-hydroxymelatonin may be a metabolite of melatonin. Overall, our data demonstrate that melatonin synthesis occurs in parallel with melatonin degradation in both chloroplasts and cytoplasm, and the resulting melatonin metabolite 2-hydroxymelatonin also acts as a signaling molecule for defense gene induction.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis catalyzing the conversion of serotonin into N-acetylserotonin. In plants, SNAT is encoded by 2 isogenes of which SNAT1 is constitutively expressed and its overexpression confers increased yield in rice. However, the role of SNAT2 remains to be clarified. In contrast to SNAT1, the diurnal rhythm of SNAT2 mRNA expression peaks at night. In this study, transgenic rice plants in which SNAT2 expression were suppressed by RNAi technology showed a decrease in melatonin and a dwarf phenotype with erect leaves, reminiscent of brassinosteroids (BR)-deficient mutants. Of note, the dwarf phenotype was dependent on the presence of dark, suggesting that melatonin is involved in dark growth (skotomorphogenesis). In support of this suggestion, SNAT2 RNAi lines exhibited photomorphogenic phenotypes such as inhibition of internodes and increased expression of light-inducible CAB genes in the dark. The causative gene for the melatonin-mediated BR biosynthetic gene was DWARF4, a rate-limiting BR biosynthetic gene. Exogenous melatonin treatment induced several BR biosynthetic genes, including DWARF4, D11, and RAVL1. As expected from the erect leaves, the SNAT2 RNAi lines produced less BR than the wild type. Our results show for the first time that melatonin is a positive regulator of dark growth or shade outgrowth by regulating BR biosynthesis in plants.

The activation of rabbit aortic smooth muscle was studied by two most widely used vascular smooth muscle stimulants: alpha-adrenoceptor activation by norepinephrine (NE) and high-K+ depolarization. This was studied by measurements of isometric contractions and net as well as unidirectional Ca2+ fluxes. These parameters showed markedly differential sensitivities towards tow smooth muscle inhibitors used in this study: D600 and amrinone. By choosing an appropriate concentration of D600 or amrinone, Ca2+ uptake or Ca2+ influx induced by high K+ or NE could by selectively inhibited. Furthermore, by using unidirectional flux measurements it was demonstrated that Ca2+ influx stimulated by NE and high K+ were additive in nature. The data from the addivity experiment exclude the interpretation of a common Ca2+ pathway with two separate mechanisms for opening it. The data on three criteria employed in this study provide evidence for the existence of two independent Ca2+ pathways, one for each mode of activation, for Ca2+ influx known to be associated with these contractions.

Melatonin has long been recognized as a positive signaling molecule and potent antioxidant in plants, which alleviates damage caused by adverse conditions such as salt, cold, and heat stress. In this study, we found a paradoxical role for melatonin in abiotic stress responses. Suppression of the serotonin N-acetyltransferase 2 (snat2) gene encoding the penultimate enzyme in melatonin biosynthesis led to simultaneous decreases in both melatonin and brassinosteroid (BR) levels, causing a semi-dwarf with erect leaf phenotype, typical of BR deficiency. Here, we further characterized snat2 rice in terms of grain morphology and abiotic stress tolerance, to determine whether snat2 rice exhibited characteristics similar to those of BR-deficient rice. As expected, the snat2 rice exhibited tolerance to multiple stress conditions including cadmium, salt, cold, and heat, as evidenced by decreased malondialdehyde (MDA) levels and increased chlorophyll levels, in contrast with SNAT2 overexpression lines, which were less tolerant to stress than wild type plants. In addition, the length and width of grain from snat2 plants were reduced relative to the wild type, which is reminiscent of BR deficiency in rice. Other melatonin-deficient mutant rice lines with suppressed BR synthesis (i.e., comt and t5h) also showed tolerance to salt and heat stress, whereas melatonin-deficient rice seedlings without decreased BR levels (i.e., tdc) failed to exhibit increased stress tolerance, suggesting that stress tolerance was increased not by melatonin deficiency alone, but by a melatonin deficiency-mediated decrease in BR.

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Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthetic pathway, in which serotonin is converted into N-acetylserotonin (NAS) in plants. To date, two SNAT isogenes with low amino acid sequence homologies have been identified. Their single suppression in rice has been reported, but their double suppression in rice has not yet been attempted. Here, we generated double-suppression transgenic rice (snat1+2) using the RNA interference technique. The snat1+2 exhibited retarded seedling growths in conjunction with severe decreases in melatonin compared to wild-types and single-suppression rice plants (snat1 or snat2). The laminar angle was decreased in the snat1+2 rice compared to that of the wild-types and snat1, but was comparable to that of snat2. The reduced germination speed in the snat1+2 was comparable to that of snat2. Seed-aging testing revealed that snat1 was the most severely deteriorated, followed by snat1+2 and snat2, suggesting that melatonin is positively involved in seed longevity.

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