Escherichia coli RimI Encodes Serotonin N-Acetyltransferase Activity and Its Overexpression Leads to Enhanced Growth and Melatonin Biosynthesis.

Serotonin N-acetyltransferase (SNAT) functions as the penultimate or final enzyme in melatonin biosynthesis, depending on the substrate. The Escherichia coli orthologue of archaeal SNAT from Thermoplasma volcanium was identified as RimI (EcRimI), with 42% amino acid similarity to archaeal SNAT. EcRimI has been reported to be an N-acetyltransferase enzyme. Here, we investigated whether EcRimI also exhibits SNAT enzyme activity. To achieve this goal, we purified recombinant EcRimI and examined its SNAT enzyme kinetics. As expected, EcRimI showed SNAT activity toward various amine substrates including serotonin and 5-methoxytryptamine, with Km and Vmax values of 531 µM and 528 pmol/min/mg protein toward serotonin and 201 µM and 587 pmol/min/mg protein toward 5-methoxytryptamine, respectively. In contrast to the rimI mutant E. coli strain that showed no growth defect, the EcRimI overexpression strain exhibited a 2-fold higher growth rate than the control strain after 24 h incubation in nutrient-rich medium. The EcRimI overexpression strain produced more melatonin than the control strain in the presence of 5-methoxytryptamine. The enhanced growth effect of EcRimI overexpression was also observed under cadmium stress. The higher growth rate associated with EcRimI expression was attributed to increased protein N-acetyltransferase activity, increased synthesis of melatonin, or the combined effects of both.

Human Naa50 Shows Serotonin N-Acetyltransferase Activity, and Its Overexpression Enhances Melatonin Biosynthesis, Resulting in Osmotic Stress Tolerance in Rice.

A new clade of serotonin N-acetyltransferase (SNAT), the penultimate enzyme in the melatonin biosynthetic pathway, has been reported in the archaeon Thermoplasma volcanium. The closest homolog of archaea SNAT in human was an N-alpha-acetyltransferase50 (Naa50). To determine whether human Naa50 (hNaa50) shows SNAT enzyme activity, we chemically synthesized and expressed the hNaa50 gene in Escherichia coli, followed by Ni2+ affinity purification. Purified recombinant hNaa50 showed SNAT activity (Km and Vmax values of 986 µM and 1800 pmol/min/mg protein, respectively). To assess its in vivo function, hNaa50 was overexpressed in rice (hNaa50-OE). The transgenic rice plants produced more melatonin than nontransgenic wild-type rice, indicating that hNaa50 is functionally coupled with melatonin biosynthesis. Due to its overproduction of melatonin, hNaa50-OE had a higher tolerance against osmotic stress than the wild type. Enhanced expression of the chaperone genes BIP1 and CNX in hNaa50-OE plants was responsible for the increased tolerance. It is concluded that hNaa50 harbors serotonin N-acetyltransferase enzyme activity in addition to its initial N-alpha-acetyltransferase, suggesting the bifunctionality of the hNaa50 enzyme toward serotonin and protein substrates. Consequently, ectopic overexpression of hNaa50 in rice enhanced melatonin synthesis, indicating that hNaa50 is in fact involved in melatonin biosynthesis.

Functional Characterization of Arylalkylamine N-Acetyltransferase, a Pivotal Gene in Antioxidant Melatonin Biosynthesis from Chlamydomonas reinhardtii.

Arylalkylamine N-acetyltransferase (AANAT) is a pivotal enzyme in melatonin biosynthesis that catalyzes the conversion of serotonin to N-acetylserotonin. Homologs of animal AANAT genes are present in animals, but not in plants. An AANAT homolog was found in Chlamydomonas reinhardtii, but not other green algae. The characteristics of C. reinhardtii AANAT (CrAANAT) are unclear. Here, full-length CrAANAT was chemically synthesized and expressed in Escherichia coli. Recombinant CrAANAT exhibited AANAT activity with a Km of 247 µM and Vmax of 325 pmol/min/mg protein with serotonin as the substrate. CrAANAT was localized to the cytoplasm in tobacco leaf cells. Transgenic rice plants overexpressing CrAANAT (CrAANAT-OE) exhibited increased melatonin production. CrAANAT-OE plants showed a longer seed length and larger second leaf angle than wild-type plants, indicative of the involvement of brassinosteroids (BRs). As expected, BR biosynthesis- and signaling-related genes such as D2, DWARF4, DWARF11, and BZR1 were upregulated in CrAANAT-OE plants. Therefore, an increased endogenous melatonin level by ectopic overexpression of CrAANAT seems to be closely associated with BR biosynthesis, thereby influencing seed size.

The Antioxidant Cyclic 3-Hydroxymelatonin Promotes the Growth and Flowering of Arabidopsis thaliana.

In plants, melatonin is metabolized into several compounds, including the potent antioxidant cyclic 3-hydroxymelatonin (3-OHM). Melatonin 3-hydroxylase (M3H), a member of the 2-oxo-glutarate-dependent enzyme family, is responsible for 3-OHM biosynthesis. Although rice M3H has been cloned, its roles are unclear, and no homologs in other plant species have been characterized. Here, we cloned and characterized Arabidopsis thaliana M3H (AtM3H). The purified recombinant AtM3H exhibited Km and Vmax values of 100 µM and 20.7 nmol/min/mg protein, respectively. M3H was localized to the cytoplasm, and its expression peaked at night. Based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, 3-OHM exhibited 15-fold higher antioxidant activity than melatonin. An Arabidopsis M3H knockout mutant (m3h) produced less 3-OHM than the wildtype (WT), thus reducing antioxidant activity and biomass and delaying flowering. These defects were caused by reduced expression of FLOWERING LOCUS T (FT) and gibberellin-related genes, which are responsible for flowering and growth. Exogenous 3-OHM, but not exogenous melatonin, induced FT expression. The peak of M3H expression at night matched the FT expression pattern. The WT and m3h exhibited similar responses to salt stress and pathogens. Collectively, our findings indicate that 3-OHM promotes growth and flowering in Arabidopsis.

Molecular Regulation of Antioxidant Melatonin Biosynthesis by Brassinosteroid Acting as an Endogenous Elicitor of Melatonin Induction in Rice Seedlings.

Gibberellic acid (GA) was recently shown to induce melatonin synthesis in rice. Here, we examined whether brassinosteroids (BRs) also induce melatonin synthesis because BRs and GA show redundancy in many functions. Among several plant hormones, exogenous BR treatment induced melatonin synthesis by twofold compared to control treatment, whereas ethylene, 6-benzylaminopurine (BA), and indole-3-acetic acid (IAA) showed negligible effects on melatonin synthesis. Correspondingly, BR treatment also induced a number of melatonin biosynthetic genes in conjunction with the suppression of melatonin catabolic gene expression. Several transgenic rice plants with downregulated BR biosynthesis-related genes, such as DWARF4, DWARF11, and RAV-Like1 (RAVL1), were generated and exhibited decreased melatonin synthesis, indicating that BRs act as endogenous elicitors of melatonin synthesis. Notably, treatment with either GA or BR fully restored melatonin synthesis in the presence of paclobutrazol, a GA biosynthesis inhibitor. Moreover, exogenous BR treatment partially restored melatonin synthesis in both RAVL1 and Gα RNAi transgenic rice plants, whereas GA treatment fully restored melatonin synthesis comparable to wild type in RAVL1 RNAi plants. Taken together, our results highlight a role of BR as an endogenous elicitor of melatonin synthesis in a GA-independent manner in rice plants.

2-Hydroxymelatonin Promotes Seed Germination by Increasing Reactive Oxygen Species Production and Gibberellin Synthesis in Arabidopsis thaliana.

It was recently reported that 2-hydroxymelatonin (2-OHM) is responsible for inducing reactive oxygen species (ROS) in plants. ROS are crucial molecules that promote germination through interaction with hormones such as gibberellic acid (GA). In this study, to confirm the pro-oxidant role of 2-OHM, we investigated its effect on seed germination in Arabidopsis thaliana (L.) Heynh. Columbia-0. We found that 2-OHM treatment stimulated seed germination by 90% and 330% in non-dormant and dormant seeds, respectively, whereas melatonin marginally increased germination (~13%) in both seed types compared to untreated control seeds. The germination promotion effects of exogenous 2-OHM treatment were due to increased ROS production followed by the induction of GA synthesis and expression of responsive genes. Accordingly, melatonin 2-hydroxylase (M2H), the gene responsible for 2-OHM synthesis, was strictly expressed only during the germination process. Further molecular genetic analyses using m2h knockout mutant and M2H overexpression clearly supported an increase in ROS triggered by 2-OHM, followed by increased expression of GA-related genes, which shortened the time to germination. Notably, 2-OHM application to m2h knockout mutant seeds fully recovered germination to levels comparable to that of the wild type, whereas melatonin treatment failed to increase germination. Together, these results indicate that 2-OHM is a pivotal molecule that triggers increased ROS production during seed germination, thereby enhancing germination via the GA pathway in Arabidopsis thaliana.

Functional Characterization of Serotonin N-Acetyltransferase in Archaeon Thermoplasma volcanium.

Serotonin N-acetyltransferase is the penultimate enzyme in the melatonin biosynthetic pathway that catalyzes serotonin into N-acetylserotonin. Many SNAT genes have been cloned and characterized from organisms ranging from bacteria to plants and mammals. However, to date, no SNAT gene has been identified from Archaea. In this study, three archaeal SNAT candidate genes were synthesized and expressed in Escherichia coli, and SNAT enzyme activity was measured using their purified recombinant proteins. Two SNAT candidate genes, from Methanoregulaceae (Archaea) and Pyrococcus furiosus, showed no SNAT enzyme activity, whereas a SNAT candidate gene from Thermoplasma volcanium previously named TvArd1 exhibited SNAT enzyme activity. The substrate affinity and the maximum reaction rate of TvSNAT toward serotonin were 621 µM and 416 pmol/min/mg protein, respectively. The highest amine substrate was tyramine, followed by tryptamine, serotonin, and 5-methoxytryptamine, which were similar to those of plant SNAT enzymes. Homologs of TvSNAT were found in many Archaea families. Ectopic overexpression of TvSNAT in rice resulted in increased melatonin content, antioxidant activity, and seed size in conjunction with the enhanced expression of seed size-related gene. This study is the first to report the discovery of SNAT gene in Archaea. Future research avenues include the cloning of TvSNAT orthologs in different phyla, and identification of their regulation and functions related to melatonin biosynthesis in living organisms.

Phytomelatonin as a signaling molecule for protein quality control via chaperone, autophagy, and ubiquitin-proteasome systems in plants.

Physiological effects mediated by melatonin are attributable to its potent antioxidant activity as well as its role as a signaling molecule in inducing a vast array of melatonin-mediated genes. Here, we propose melatonin as a signaling molecule essential for protein quality control (PQC) in plants. PQC occurs by the coordinated activities of three systems: the chaperone network, autophagy, and the ubiquitin-proteasome system. With regard to the melatonin-mediated chaperone pathway, melatonin increases thermotolerance by induction of heat shock proteins and confers endoplasmic reticulum stress tolerance by increasing endoplasmic reticulum chaperone proteins. In chloroplasts, melatonin-induced chaperones, including Clps and CpHSP70s, play key roles in the PQC of chloroplast-localized proteins, such as Lhcb1, Lhcb4, and RBCL, during growth. Melatonin regulates PQC by autophagy processes, in which melatonin induces many autophagy (ATG) genes and autophagosome formation under stress conditions. Finally, melatonin-mediated plant stress tolerance is associated with up-regulation of stress-induced transcription factors, which are regulated by the ubiquitin-proteasome system. In this review, we propose that melatonin plays a pivotal role in PQC and consequently functions as a pleiotropic molecule under non-stress and adverse conditions in plants.

Exogenous Gibberellin Treatment Enhances Melatonin Synthesis for Melatonin-Enriched Rice Production.

Melatonin production is induced by many abiotic and biotic stressors; it modulates the levels of many plant hormones and their signaling pathways. This study investigated the effects of plant hormones on melatonin synthesis. Melatonin synthesis in rice seedlings was significantly induced upon exogenous gibberellin 3 (GA3) treatment, while it was severely decreased by GA synthesis inhibitor paclobutrazol. In contrast, abscisic acid (ABA) strongly inhibited melatonin synthesis, whereas its inhibitor norflurazon (NF) induced melatonin synthesis. The observed GA-mediated increase in melatonin was closely associated with elevated expression levels of melatonin biosynthetic genes such as TDC3, T5H, and ASMT1; it was also associated with reduced expression levels of catabolic genes ASDAC and M2H. In a paddy field, the treatment of immature rice seeds with exogenous GA led to enhanced melatonin production in rice seeds; various transgenic rice plants downregulating a GA biosynthesis gene (GA3ox2) and a signaling gene (Gα) showed severely decreased melatonin levels, providing in vivo genetic evidence that GA has a positive effect on melatonin synthesis. This is the first study to report that GA is positively involved in melatonin synthesis in plants; GA treatment can be used to produce melatonin-rich seeds, vegetables, and fruits, which are beneficial for human health.

2-Hydroxymelatonin, Rather Than Melatonin, Is Responsible for RBOH-Dependent Reactive Oxygen Species Production Leading to Premature Senescence in Plants.

Unlike animals, plants amply convert melatonin into 2-hydroxymelatonin (2-OHM) and cyclic 3-hydroxymelatonin (3-OHM) through the action of melatonin 2-hydroxylase (M2H) and melatonin 3-hydroxylase (M3H), respectively. Thus, the effects of exogenous melatonin treatment in plants may be caused by melatonin, 2-OHM, or 3-OHM, or some combination of these compounds. Indeed, studies of melatonin's effects on reactive oxygen species (ROS) production have reported conflicting results. In this study, we demonstrated that 2-OHM treatment induced ROS production, whereas melatonin did not. ROS production from 2-OHM treatment occurred in old arabidopsis leaves in darkness, consistent with an ethylene-mediated senescence mechanism. Transgenic tobacco plants containing overexpressed rice M2H exhibited dwarfism and leaf necrosis of the upper leaves and early senescence of the lower leaves. We also demonstrated that 2-OHM-mediated ROS production is respiratory burst NADPH oxidase (RBOH)-dependent and that 2-OHM-induced senescence genes require ethylene and the abscisic acid (ABA) signaling pathway in arabidopsis. In contrast to melatonin, 2-OHM treatment induced senescence symptoms such as leaf chlorosis and increased ion leakage in arabidopsis. Senescence induction is known to begin with decreased levels of proteins involved in chloroplast maintenance, including Lhcb1 and ClpR1. Together, these results show that 2-OHM acts as a senescence-inducing factor by inducing ROS production in plants.

Inhibition of Rice Serotonin N-Acetyltransferases by MG149 Decreased Melatonin Synthesis in Rice Seedlings.

We examined the effects of two histone acetyltransferase (HAT) inhibitors on the activity of rice serotonin N-acetyltransferases (SNAT). Two rice recombinant SNAT isoenzymes (SNAT1 and SNAT2) were incubated in the presence of either MG149 or MB3, HAT inhibitors. MG149 significantly inhibited the SNAT enzymes in a dose-dependent manner, especially SNAT1, while SNAT2 was moderately inhibited. By contrast, MB3 had no effect on SNAT1 or SNAT2. The application of 100 µM MG149 to rice seedlings decreased melatonin by 1.6-fold compared to the control, whereas MB3 treatment did not alter the melatonin level. MG149 significantly decreased both melatonin and N-acetylserotonin when rice seedlings were challenged with cadmium, a potent elicitor of melatonin synthesis in rice. Although MG149 inhibited melatonin synthesis in rice seedlings, no melatonin deficiency-induced lamina angle decrease was observed due to the insufficient suppression of SNAT2, which is responsible for the lamina angle decrease in rice.

Melatonin Regulates Chloroplast Protein Quality Control via a Mitogen-Activated Protein Kinase Signaling Pathway.

Serotonin N-acetyltransferase 1 (SNAT1), the penultimate enzyme for melatonin biosynthesis has shown N-acetyltransferase activity toward multiple substrates, including histones, serotonin, and plastid proteins. Under two different light conditions such as 50 or 100 µmol m-2 s-1, a SNAT1-knockout (snat1) mutant of Arabidopsis thaliana ecotype Columbia (Col-0) exhibited small size phenotypes relative over wild-type (WT) Arabidopsis Col-0. Of note, the small phenotype is stronger when growing at the 50 µmol m-2 s-1, exhibiting a dwarfism phenotype and delayed flowering. The snat1 Arabidopsis Col-0 accumulated less starch than the WT Col-0. Moreover, snat1 exhibited lower Lhcb1, Lhcb4, and RBCL protein levels, compared with the WT Col-0, but no changes in the corresponding transcripts, suggesting the involvement of melatonin in chloroplast protein quality control (CPQC). Accordingly, caseinolytic protease (Clp) and chloroplast heat shock proteins (CpHSPs), two key proteins involved in CPQC, as well as ROS defense were suppressed in snat1. In contrast, exogenous melatonin treatment induced expression of Clp, CpHSP, APX1, and GST, but not other growth-related genes such as DWF4, KS, and IAA1. Finally, the induction of ClpR1, APX1, and GST1 in response to melatonin was inhibited in the mitogen-activated protein kinase (MAPK) knockdown Arabidopsis (mpk3/6), suggesting that melatonin-mediated CPQC was mediated, in part, by the MAPK signaling cascade. These results suggest that melatonin is involved in CPQC, which plays a pivotal role in starch synthesis in plants.

Suppression of Rice Cryptochrome 1b Decreases Both Melatonin and Expression of Brassinosteroid Biosynthetic Genes Resulting in Salt Tolerance.

We investigated the relationship between the blue-light photoreceptor cryptochrome (CRY) and melatonin biosynthesis by generating RNA interference (RNAi) transgenic rice plants that suppress the cryptochrome 1b gene (CRY1b). The resulting CRY1b RNAi rice lines expressed less CRY1b mRNA, but not CRY1a or CRY2 mRNA, suggesting that the suppression is specific to CRY1b. The growth of CRY1b RNAi rice seedlings was enhanced under blue light compared to wild-type growth, providing phenotypic evidence for impaired CRY function. When these CRY1b RNAi rice plants were challenged with cadmium to induce melatonin, wild-type plants produced 100 ng/g fresh weight (FW) melatonin, whereas CRY1b RNAi lines produced 60 ng/g FW melatonin on average, indicating that melatonin biosynthesis requires the CRY photoreceptor. Due to possible feedback regulation, the expression of melatonin biosynthesis genes such as T5H, SNAT1, SNAT2, and COMT was elevated in the CRY1b RNAi lines compared to the wild-type plants. In addition, laminar angles decreased in the CRY1b RNAi lines via the suppression of brassinosteroid (BR) biosynthesis genes such as DWARF. The main cause of the BR decrease in the CRY1b RNAi lines seems to be the suppression of CRY rather than decreased melatonin because the melatonin decrease suppressed DWARF4 rather than DWARF.

Melatonin metabolism, signaling and possible roles in plants.

Melatonin is a multifunctional biomolecule found in both animals and plants. In this review, the biosynthesis, levels, signaling, and possible roles of melatonin and its metabolites in plants is summarized. Tryptamine 5-hydroxylase (T5H), which catalyzes the conversion of tryptamine into serotonin, has been proposed as a target to create a melatonin knockout mutant presenting a lesion-mimic phenotype in rice. With a reduced anabolic capacity for melatonin biosynthesis and an increased catabolic capacity for melatonin metabolism, all plants generally maintain low melatonin levels. Some plants, including Arabidopsis and Nicotiana tabacum (tobacco), do not possess tryptophan decarboxylase (TDC), the first committed step enzyme required for melatonin biosynthesis. Major melatonin metabolites include cyclic 3-hydroxymelatonin (3-OHM) and 2-hydroxymelatonin (2-OHM). Other melatonin metabolites such as N1 -acetyl-N2 -formyl-5-methoxykynuramine (AFMK), N-acetyl-5-methoxykynuramine (AMK) and 5-methoxytryptamine (5-MT) are also produced when melatonin is applied to Oryza sativa (rice). The signaling pathways of melatonin and its metabolites act via the mitogen-activated protein kinase (MAPK) cascade, possibly with Cand2 acting as a melatonin receptor, although the integrity of Cand2 remains controversial. Melatonin mediates many important functions in growth stimulation and stress tolerance through its potent antioxidant activity and function in activating the MAPK cascade. The concentration distribution of melatonin metabolites appears to be species specific because corresponding enzymes such as M2H, M3H, catalases, indoleamine 2,3-dioxygenase (IDO) and N-acetylserotonin deacetylase (ASDAC) are differentially expressed among plant species and even among different tissues within species. Differential levels of melatonin and its metabolites can lead to differential physiological effects among plants when melatonin is either applied exogenously or overproduced through ectopic overexpression.

Effects of Light Quality and Phytochrome Form on Melatonin Biosynthesis in Rice.

Light is an important factor influencing melatonin synthesis in response to cadmium treatment in rice. However, the effects of light quality on, and the involvement of phytochrome light receptors in, melatonin production have not been explored. In this study, we used light-emitting diodes (LEDs) to investigate the effect of light wavelength on melatonin synthesis, and the role of phytochromes in light-dependent melatonin induction in rice. Upon cadmium treatment, peak melatonin production was observed under combined red and blue (R + B) light, followed by red (R) and blue light (B). However, both far-red (FR) LED light and dark treatment (D) failed to induce melatonin production. Similarly, rice seedlings grown under the R + B treatment showed the highest melatonin synthesis, followed by those grown under B and R. These findings were consistent with the results of our cadmium treatment experiment. To further confirm the effects of light quality on melatonin synthesis, we employed rice photoreceptor mutants lacking functional phytochrome genes. Melatonin induction was most inhibited in the phytochrome A mutant (phyA) followed by the phyB mutant under R + B treatment, whereas phyB produced the least amount of melatonin under R treatment. These results indicate that PhyB is an R light receptor. Expression analyses of genes involved in melatonin biosynthesis clearly demonstrated that tryptophan decarboxylase (TDC) played a key role in phytochrome-mediated melatonin induction when rice seedlings were challenged with cadmium.

Simultaneous Suppression of Two Distinct Serotonin N-Acetyltransferase Isogenes by RNA Interference Leads to Severe Decreases in Melatonin and Accelerated Seed Deterioration in Rice.

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.

Knockout of Arabidopsis Serotonin N-Acetyltransferase-2 Reduces Melatonin Levels and Delays Flowering.

Melatonin plays roles in both plant growth and defense. Serotonin N-acetyltransferase (SNAT) catalyzes formation of N-acetylserotonin (NAS) from serotonin. Plants contain two SNAT isogenes, which exhibit low-level amino acid homology. We studied the ArabidopsisthalianaSNAT2 (AtSNAT2) gene; we prepared recombinant SNAT2 protein and characterized a snat2 knockout mutant. The SNAT2 protein exhibited 27% amino acid homology with SNAT1; the Km was 232 µM and the Vmax was 2160 pmol/min/mg protein. Melatonin inhibited SNAT enzyme activity in vitro. SNAT2 mRNA was abundantly expressed in flowers; the melatonin content of flowers of the snat2 mutant was significantly less than that of wild-type flowers. The mutant exhibited delayed flowering and reductions in leaf area and biomass compared to the wild type. Delayed flowering was attributable to reductions in the expression levels of the gibberellin biosynthetic genes ent-kaurene synthase (KS) and FLOWERING LOCUS T (FT).

Suppression of Melatonin 2-Hydroxylase Increases Melatonin Production Leading to the Enhanced Abiotic Stress Tolerance against Cadmium, Senescence, Salt, and Tunicamycin in Rice Plants.

Melatonin 2-hydroxylase (M2H) catalyzes the conversion of melatonin into 2hydroxymelatonin (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 3hydroxymelatonin 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.

Melatonin Deficiency Confers Tolerance to Multiple Abiotic Stresses in Rice via Decreased Brassinosteroid Levels.

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.

Melatonin-deficient rice plants show a common semidwarf phenotype either dependent or independent of brassinosteroid biosynthesis.

Melatonin-deficient rice with a semidwarf erect-leaf phenotype was created by suppressing serotonin N-acetyltransferase 2 (SNAT2). We generated an RNAi transgenic rice that suppressed tryptophan decarboxylase (TDC), which encodes the first TDC enzyme committed step for melatonin biosynthesis in plants catalyzing the conversion of tryptophan into tryptamine, to determine whether other transgenic rice with downregulated melatonin biosynthetic genes exhibited the same erect-leaf phenotype as the snat2 RNAi rice. The TDC RNAi rice produced significantly less melatonin than the wild type and exhibited a semidwarf phenotype, but no erect-leaf phenotype was observed. In contrast, tryptamine 5-hydroxylase (T5H) knockout Sekiguchi rice and caffeic acid O-methyltransferase (COMT) RNAi rice seedlings were semidwarf phenotypes with erect leaves, as was the snat2 RNAi rice due to a melatonin deficiency. All RNAi rice plants showing erect-leaf phenotypes had lower expression levels of the DWARF4 gene, which is a key enzyme for brassinosteroid (BR) biosynthesis, leading to lower BR levels than their respective wild types. Suppressing melatonin synthesis did not alter the contents of indole 3-acetic acid (IAA), suggesting the irrelevance of melatonin deficiency to IAA biosynthesis. These data indicate that a semidwarf seedling is a common rice phenotype by the lack of melatonin synthesis with or without BR suppression in a melatonin biosynthetic gene-specific manner.