Molecular cloning of a plant N-acetylserotonin methyltransferase and its expression characteristics in rice

N-acetylserotonin methyltransferase (ASMT), the last enzyme in the synthesis of melatonin, catalyzes N-acetylserotonin into melatonin. For the first time, we cloned ASMT from rice through the analysis of recombinant Escherichia coli harboring putative rice O-methyltransferase (OMT) cDNAs. In total, 18 full-length cDNAs, which show homology to wheat caffeic acid 3-O-methyltransferase, were expressed in E. coli and induced in the presence of N-acetylserotonin; we then analyzed the production of melatonin. Only recombinant E. coli line 15 showed melatonin synthesis; no other recombinant lines produced melatonin with the addition of N-acetylserotonin in E. coli culture. Line 15 clearly exhibited in vitro ASMT enzyme activity with 0.27 pkat/mg protein. ASMT enzyme activity was inhibited by various related compounds such as N-acetyltryptamine and N-acetyltyrosine. The open reading frame of ASMT consists of 364 amino acids possessing well-conserved motifs found in plant OMT such as S-adenosyl-L-methionine-binding and catalytic sites. Induction patterns of ASMT mRNA were well matched with the production of melatonin in rice leaves during senescence, as well as several stressors.     

Light-regulated melatonin biosynthesis in rice during the senescence process in detached leaves

The effect of light on melatonin biosynthesis was examined in detached rice (Oryza sativa cv. Asahi) leaves during the senescence process. The detached leaves were exposed to senescence treatment either in constant darkness or in constant light, and subjected to HPLC analysis for melatonin and its precursors. Higher melatonin levels were detected in rice leaves under constant light while very low levels were observed in constant darkness. Levels of the melatonin intermediates, tryptamine, serotonin, and N-acetylserotonin significantly decreased in the dark compared to those in the light. Furthermore, relative mRNA levels of melatonin biosynthetic genes and their corresponding proteins decreased accordingly in constant darkness. The most striking difference between constant light and dark was observed in levels of the protein tryptamine 5-hydroxylase. These results suggest that melatonin biosynthesis during senescence is dependent on light signals in rice leaves, contrary to the response found in animals.     

Elevated production of melatonin in transgenic rice seeds expressing rice tryptophan decarboxylase

A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well‐known bioactive molecule with an array of health‐promoting properties, including potent antioxidant capability. To generate melatonin‐rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3‐1) had melatonin concentrations 31‐fold higher than those of wild‐type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild‐type levels. Thus, a seed‐specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5‐hydroxytryptophan, tryptamine, serotonin, and N‐acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene.     

Caffeic acid O‐methyltransferase is involved in the synthesis of melatonin by methylating N‐acetylserotonin in Arabidopsis

Although a plant N‐acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. To clone an ASMT de novo from a dicotyledonous plant, we expressed eight Arabidopsis thaliana O‐methyltransferase (OMT) cDNAs in Escherichia coli and screened for ASMT activity by measuring melatonin production after the application of 1 mm N‐acetylserotonin (NAS). Among the eight strains harboring the full‐length cDNAs, the OMT3 strain produced high levels of melatonin, suggesting that OMT3 encodes an active ASMT. OMT3 is already known as caffeic acid OMT (COMT), suggesting multiple functions for this enzyme. The purified recombinant A. thaliana COMT (AtCOMT) showed high ASMT activity, catalyzing the conversion of NAS to melatonin. The K_m and V_max values for ASMT activity were 233 μm and 1800 pmol/min/mg protein, while the K_m and V_max values for COMT activity were 103 μm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) for ASMT activity was 709‐fold lower than for COMT. In vitro, ASMT activity was dramatically decreased by the addition of caffeic acid in a dose‐dependent manner, but the activity of COMT was not altered by NAS. Lastly, the Arabidopsis comt knockout mutant exhibited less production of melatonin than the wild type when Arabidopsis leaves were infiltrated with 1 mm NAS, suggestive of in vivo role of COMT in melatonin biosynthesis in plants.     

Molecular cloning of rice serotonin N‐acetyltransferase,the penultimate gene in plant melatonin biosynthesis

Because of the absence of an arylalkylamine N‐acetyltransferase (AANAT) homolog in the plant genome, the proposal was made that a GCN5‐related N‐acetyltransferase superfamily gene (GNAT) could be substituted for AANAT. To clone rice serotonin N‐acetyltransferase (SNAT), we expressed 31 rice GNAT cDNAs in Escherichia coli and screened SNAT activity by measuring N‐acetyltryptamine after application with 1 mm tryptamine. GNAT5 was shown to produce high levels of N‐acetyltryptamine in E. coli, suggesting a possible rice SNAT. To confirm SNAT activity, the GNAT5 protein was purified through affinity purification from E. coli culture. The purified recombinant GNAT5 showed high SNAT enzyme activity catalyzing serotonin into N‐acetylserotonin. The values for K_m and V_max were 385 μm and 282 pmol/min/mg protein, respectively. An in vitro enzyme assay of purified SNAT showed N‐acetylserotonin formation to be proportional to enzyme concentration and time, with peak activity at pH 8.8. High substrate concentrations above 1 mm serotonin inhibited SNAT activity. Finally, the mRNA level of SNAT was higher in shoots than in roots, but it was expressed constitutively, unlike N‐acetylserotonin methyltransferase (ASMT), the terminal enzyme in melatonin synthesis. These results suggest that ASMT rather than SNAT is the rate‐limiting enzyme of melatonin biosynthesis in plants.     

Enhanced production of melatonin by ectopic overexpression of human serotonin N‐acetyltransferase plays a role in cold resistance in transgenic rice seedlings

Abstract: Serotonin N‐acetyltransferase (SNA), a rate‐limiting enzyme in melatonin biosynthesis in vertebrates, is responsible for the production of N‐acetylserotonin; this molecule is then converted to melatonin by hydroxyindole‐O‐methyltransferase. We generated transgenic rice plants via expression of the human SNA gene under the constitutive ubiquitin promoter using Agrobacterium‐mediated gene transformation. We investigated the role of SNA in the biosynthesis of melatonin and the physiological role of melatonin in rice plants. The integration and expression of the transgene were confirmed in T₁ transgenic rice seedlings by Southern, Northern, and RT‐PCR analyses. High SNA‐specific enzyme activities were observed in the transgenic rice plants, whereas the wild type revealed a trace level of SNA enzyme activity. The functional expression of SNA protein was closely associated with the elevated synthesis of N‐acetylserotonin and melatonin in the transgenic rice plants. Experiments using both exogenous treatment of serotonin and senescent detached leaves, which contain a pool of serotonin, significantly enhanced melatonin biosynthesis, indicating that endogenous serotonin levels play a bottleneck role in the pathway of melatonin biosynthesis. Finally, the transgenic rice seedlings with high levels of melatonin showed elevated chlorophyll synthesis during cold stress, suggesting a role for melatonin in cold‐stress resistance.     

Low melatonin production by suppression of either serotonin N‐acetyltransferase or N‐acetylserotonin methyltransferase in rice causes seedling growth retardation with yield penalty,abiotic stress susceptibility,and enhanced coleoptile growth under anoxic conditions

Serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT) are the last two key enzymes for melatonin biosynthesis in living organisms. In this study, we demonstrated that transgenic rice (Oryza sativa L.) plants, in which expression of either endogenous SNAT or ASMT was suppressed, had reduced melatonin synthesis, confirming that both SNAT and ASMT are functionally involved in melatonin synthesis. The melatonin‐deficient SNAT rice had retarded seedling growth, which was partially restored by exogenous melatonin application, suggesting melatonin's role in seedling growth. In addition, the plants were more sensitive to various abiotic stresses, including salt and cold, compared with the wild type. Melatonin‐deficient SNAT rice had increased coleoptile growth under anoxic conditions, indicating that melatonin also inversely regulates plant growth under anaerobic conditions with the concomitant high expression of alcohol dehydrogenase genes. Similarly, the melatonin‐deficient ASMT rice exhibited accelerated senescence in detached flag leaves, as well as significantly reduced yield. These loss‐of‐function studies on the melatonin biosynthetic genes confirmed most previous pharmacological reports that melatonin not only promotes plant growth but also mitigates various abiotic stresses.     

Chloroplast overexpression of rice caffeic acid O‐methyltransferase increases melatonin production in chloroplasts via the 5‐methoxytryptamine pathway in transgenic rice plants

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.     

Chloroplastic and cytoplasmic overexpression of sheep serotonin N‐acetyltransferase in transgenic rice plants is associated with low melatonin production despite high enzyme activity

Serotonin N‐acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N‐acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast‐targeted CTS:OaSNAT lines and cytoplasm‐expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N‐acetylserotonin, melatonin, or 2‐hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis.     

Tryptamine 5-hydroxylase-deficient Sekiguchi rice induces synthesis of 5-hydroxytryptophan and N-acetyltryptamine but decreases melatonin biosynthesis during senescence process of detached leaves

Abstract: Melatonin biosynthesis was examined in Sekiguchi mutant rice lacking functional tryptamine 5‐hydroxylase (T5H) activity, which is the terminal enzyme for serotonin biosynthesis in rice. During senescence process, the leaves of Sekiguchi mutant rice produced more tryptamine and N‐acetyltryptamine compared with the wild‐type Asahi leaves. Even though T5H activity is absent, Sekiguchi leaves produce low levels of serotonin derived from 5‐hydroxytryptophan, which was found to be synthesized during senescence process. Accordingly, both rice cultivars exhibited similar levels of N‐acetylserotonin until 6 days of senescence induction; however, only Asahi leaves continued to accumulate N‐acetylserotonin after 6 days. In contrast, a large amount of N‐acetyltryptamine was accumulated in Sekiguchi leaves, indicating that tryptamine was efficiently utilized as substrate by the rice arylalkylamine N‐acetyltransferase enzyme. An increase in N‐acetyltryptamine in Sekiguchi had an inhibitory effect on synthesis of melatonin because little melatonin was produced in Sekiguchi leaves at 6 days of senescence induction, even in the presence of equivalent levels of N‐acetylserotonin in both cultivars. The exogenous treatment of 0.1 mm N‐acetyltryptamine during senescence process completely blocked melatonin synthesis.     

Placental melatonin production and melatonin receptor expression are altered in preeclampsia: new insights into the role of this hormone in pregnancy

Abstract: The melatonin system in preeclamptic pregnancies has been largely overlooked, especially in the placenta. We have previously documented melatonin production and expression of its receptors in normal human placentas. In addition, we and others have shown a beneficial role of melatonin in placental and fetal functions. In line with this, decreased maternal blood levels of melatonin are found in preeclamptic compared with normotensive pregnancies. However, melatonin production and expression of its receptors in preeclamptic compared with normotensive pregnancy placentas has never been examined. This study compares (i) melatonin‐synthesizing enzyme expression and activity, (ii) melatonin and serotonin, melatonin’s immediate precursor, levels and (iii) expression of MT1 and MT2 melatonin receptors in placentas from preeclamptic and normotensive pregnancies. Protein and mRNA expression of aralkylamine N‐acetyltransferase (AANAT) and hydroxyindole O‐methyltransferase (HIOMT), the melatonin‐synthesizing enzymes, as well as MT1 and MT2 receptors were determined by RT‐qPCR and Western blot, respectively. The activities of melatonin‐synthesizing enzymes were assessed by radiometric assays while melatonin levels were determined by LC‐MS/MS. There is a significant inhibition of AANAT, melatonin’s rate‐limiting enzyme, expression and activity in preeclamptic placentas, correlating with decreased melatonin levels. Likewise, MT1 and MT2 expression is significantly reduced in preeclamptic compared with normotensive pregnancy placentas. We propose that reduced maternal plasma melatonin levels may be an early diagnostic tool to identify pregnancies complicated by preeclampsia. This study indicates a clinical utility of melatonin as a potential treatment for preeclampsia in women where reduced maternal plasma levels have been identified.     

A rice chloroplast transit peptide sequence does not alter the cytoplasmic localization of sheep serotonin N‐acetyltransferase expressed in transgenic rice plants

Ectopic overexpression of melatonin biosynthetic genes of animal origin has been used to generate melatonin‐rich transgenic plants to examine the functional roles of melatonin in plants. However, the subcellular localization of these proteins expressed in the transgenic plants remains unknown. We studied the localization of sheep (Ovis aries) serotonin N‐acetyltransferase (OaSNAT) and a translational fusion of a rice SNAT transit peptide to OaSNAT (TS:OaSNAT) in plants. Laser confocal microscopy analysis revealed that both OaSNAT and TS:OaSNAT proteins were localized to the cytoplasm even with the addition of the transit sequence to OaSNAT. Transgenic rice plants overexpressing the TS:OaSNAT fusion transgene exhibited high SNAT enzyme activity relative to untransformed wild‐type plants, but lower activity than transgenic rice plants expressing the wild‐type OaSNAT gene. Melatonin levels in both types of transgenic rice plant corresponded well with SNAT enzyme activity levels. The TS:OaSNAT transgenic lines exhibited increased seminal root growth relative to wild‐type plants, but less than in the OaSNAT transgenic lines, confirming that melatonin promotes root growth. Seed‐specific OaSNAT expression under the control of a rice prolamin promoter did not confer high levels of melatonin production in transgenic rice seeds compared with seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter.     

Cellular localization and kinetics of the rice melatonin biosynthetic enzymes SNAT and ASMT

Serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT) are the final two enzymes in the melatonin synthesis pathway in plants. Although their corresponding genes have been cloned, their cellular localization and enzymatic characteristics are unknown. Using confocal microscopy, we showed that SNAT protein is localized in chloroplasts, whereas ASMT is expressed in the cytoplasm. In vitro measurement of ASMT enzyme activity revealed a peak of activity in roots, but SNAT enzyme activity was not detected in any plant tissues. This may be attributed in part to an effect of chlorophyll because SNAT enzyme activity was greatly inhibited by chlorophyll in a dose‐dependent manner. Because the SNAT protein of cyanobacteria is thermophilic, we examined the effect of temperature on the activity of the rice SNAT and ASMT enzymes. Purified recombinant rice SNAT and ASMT enzymes had an optimum temperature for activity of 55°C. The K_m and V_max values for SNAT at 55°C were 270 μm and 3.3 nmol/min/mg protein, whereas the K_m and V_max for ASMT were 222 μm and 9 nmol/min/mg protein, respectively. The catalytic efficiency (V_max/K_m) values of SNAT and ASMT were 16‐fold and 4054‐fold higher at 55°C than at 30°C suggestive of increased melatonin production at high temperature in plants.     

An increase in melatonin in transgenic rice causes pleiotropic phenotypes,including enhanced seedling growth,delayed flowering,and low grain yield

No previous reports have described the effects of an increase in endogenous melatonin levels on plant yield and reproduction. Here, the phenotypes of melatonin‐rich transgenic rice plants overexpressing sheep serotonin N‐acetyltransferase were investigated under field conditions. Early seedling growth of melatonin‐rich transgenic rice was greatly accelerated, with enhanced biomass relative to the wild type (WT). However, flowering was delayed by 1 wk in the transgenic lines compared with the WT. Grain yields of the melatonin‐rich transgenic lines were reduced by 33% on average. Other phenotypes also varied among the transgenic lines. For example, the transgenic line S1 exhibited greater height and biomass than the WT, while the S10 transgenic line showed diminished height and an increase in panicle numbers per plant. The expression levels of Oryza sativa homeobox1 (OSH1) and TEOSINTE BRANCHED1 (TB1) genes, two key regulators of meristem initiation and maintenance, were not altered in the transgenic lines. These data demonstrate that an alteration of endogenous melatonin levels leads to pleiotropic effects such as height, biomass, panicle number, flowering time, and grain yield, indicating that melatonin behaves as a signaling molecule in plant growth and reproduction.