Cloning of Arabidopsis serotonin N‐acetyltransferase and its role with caffeic acid O‐methyltransferase in the biosynthesis of melatonin in vitro despite their different subcellular localizations

Serotonin N‐acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis. We cloned SNAT from Arabidopsis thaliana (AtSNAT) and functionally characterized this enzyme for the first time from dicotyledonous plants. Similar to rice SNAT, AtSNAT was found to localize to chloroplasts with peak enzyme activity at 45°C (K_m, 309 μm ; V_max, 1400 pmol/min/mg protein). AtSNAT also catalyzed 5‐methoxytryptamine (5‐MT) into melatonin with high catalytic activity (K_m, 51 μm ; V_max, 5300 pmol/min/mg protein). In contrast, Arabidopsis caffeic acid O‐methyltransferase (AtCOMT) localized to the cytoplasm. Interestingly, AtCOMT can methylate serotonin into 5‐MT with low catalytic activity (K_m, 3.396 mm ; V_max, 528 pmol/min/mg protein). These data suggest that serotonin can be converted into either N‐acetylserotonin by SNAT or into 5‐MT by COMT, after which it is metabolized into melatonin by COMT or SNAT, respectively. To support this hypothesis, serotonin was incubated in the presence of both AtSNAT and AtCOMT enzymes. In addition to melatonin production, the production of major intermediates depended on incubation temperatures; N‐acetylserotonin was predominantly produced at high temperatures (45°C), while low temperatures (37°C) favored the production of 5‐MT. Our results provide biochemical evidence for the presence of a serotonin O‐methylation pathway in plant melatonin biosynthesis.     

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.     

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.     

Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots

Abstract: Rice (Oryza sativa) N‐acetylserotonin methyltransferase (osASMT), the last enzyme in the synthesis of melatonin, was expressed in Escherichia coli and purified. We then characterized its enzyme kinetics, which is the first time this has been performed in plants. Purified glutathione S‐transferase (GST)‐fused recombinant osASMT (GST‐osASMT) and GST‐free osASMT showed specific enzyme activities of 6.6 and 12.6 pmol/min per mg protein, respectively. When evaluated by the Lineweaver‐Burk equation, GST‐free osASMT exhibited a K_m of 864 μm . An in vitro enzyme assay of purified osASMT showed melatonin formation to be proportional to the enzyme and substrate concentrations, as well as time. Unlike animal ASMT, high substrate concentrations did not inhibit the activity of osASMT. Finally, melatonin biosynthesis in rice seedlings was affected by light intensity, with etiolated shoots grown in continuous darkness producing more melatonin than shoots grown in continuous light. The level of melatonin in relation to the light intensity closely paralleled the mRNA level of osASMT in the shoots, suggesting that endogenous melatonin is upregulated in darkness, as is the case in animals.     

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.     

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.     

Rice histone deacetylase 10 and Arabidopsis histone deacetylase 14 genes encode N‐acetylserotonin deacetylase,which catalyzes conversion of N‐acetylserotonin into serotonin,a reverse reaction for melatonin biosynthesis in plants

In plants, melatonin production is strictly regulated, unlike the production of its precursor, serotonin, which is highly inducible in response to stimuli, such as senescence and pathogen exposure. Exogenous serotonin treatment does not greatly induce the production of N‐acetylserotonin (NAS) and melatonin in plants, which suggests the possible existence of one or more regulatory genes in the pathway for the biosynthesis of melatonin from serotonin. In this report, we found that NAS was rapidly and abundantly converted into serotonin in rice seedlings, indicating the presence of an N‐acetylserotonin deacetylase (ASDAC). To clone the putative ASDAC gene, we screened 4 genes that were known as histone deacetylase (HDAC) genes, but encoded proteins targeted into chloroplasts or mitochondria rather than nuclei. Of 4 recombinant Escherichia coli strains expressing these genes, one E. coli strain expressing the rice HDAC10 gene was found to be capable of producing serotonin in response to treatment with NAS. The recombinant purified rice HDAC10 (OsHDAC10) protein exhibited ASDAC enzyme activity toward NAS, N‐acetyltyramine (NAT), N‐acetyltryptamine, and melatonin, with the highest ASDAC activity for NAT. In addition, its Arabidopsis ortholog, AtHDAC14, showed similar ASDAC activity to that of OsHDAC10. Both OsHDAC10 and AtHDAC14 were found to be expressed in chloroplasts. Phylogenetic analysis indicated that ASDAC homologs were present in archaea, but not in cyanobacteria, which differs from the distribution of serotonin N‐acetyltransferase (SNAT). This suggests that SNAT and ASDAC may have evolved differently from ancestral eukaryotic cells.     

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.     

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.     

A congenic line of the C57BL/6J mouse strain that is proficient in melatonin synthesis

The C57BL/6J (B6) is the most common inbred mouse strain used in biomedical research in the United States. Yet, this strain is notoriously known for being deficient in the biosynthesis of melatonin, an important effector of circadian clocks in the brain and in the retina. Melatonin deficiency in this strain results from nonfunctional alleles of the genes coding 2 key enzymes of the melatonin synthesis pathway: arylalkylamine‐N‐acetyltransferase (Aanat) and N‐acetylserotonin‐O‐methyltransferase (Asmt). By introducing functional alleles of the Aanat and Asmt genes from the melatonin‐proficient CBA/CaJ (CBA) mouse strain to B6, we have generated a B6 congenic line that has acquired the capacity of rhythmic melatonin synthesis. In addition, the melatonin‐dependent rhythm of dopamine release in the retina is restored in the B6 congenic line. Finally, we have partially characterized the Aanat and Asmt genes of the CBA strain and have identified multiple differences between CBA and B6 alleles, including single nucleotide polymorphism and deletion/insertion of DNA segments of various sizes. As an improved model organism with functional components of the melatonin synthesis pathway and melatonin‐dependent circadian regulations, the new line will be useful to researchers studying melatonin physiological functions in a variety of fields including, but not limited to, circadian biology and neuroscience. In particular, the congenic line will be useful to speed up introduction of melatonin production capacity into genetically modified mouse lines of interest such as knockout lines, many of which are on B6 or mixed B6 backgrounds. The melatonin‐proficient B6 congenic line will be widely distributed.