Sangkyu Park

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.

Melatonin-rich transgenic rice plants exhibit resistance to herbicide-induced oxidative stress

To examine whether melatonin‐rich plants can defend against oxidative stress, we subjected melatonin‐rich transgenic (MRT) rice plants to the singlet‐oxygen‐generating herbicide butafenacil. Both MRT and transgenic control (TC; expressing the vector only) rice seeds germinated and grew equally well in continuous dark on half‐strength Murashige and Skoog (MS) medium containing 0.1 μm butafenacil. However, after transferring the seedlings to light, the TCs rapidly necrotized, whereas the MRT seedlings showed resistant phenotypes. Seven‐day‐old MRT seedlings treated with 0.1 μm butafenacil were resistant to the herbicide and contained high chlorophyll levels and low malondialdehyde and hydrogen peroxide contents compared with the TCs. As they did before the herbicide treatment, the MRT plants also produced much more melatonin after the herbicide treatment than the TCs. In addition, the MRT plants exhibited higher superoxide dismutase and catalase activities before and after the herbicide treatment compared with the TCs. This is the first report showing that MRT plants exhibit resistance against a peroxidizing herbicide that acts by generating reactive oxygen species (ROS) that kill plants. This result indicates that melatonin scavenges ROS efficiently in vivo in the transgenic plants, leading to oxidative stress resistance.

Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves.

Serotonin, which is well known as a pineal hormone in mammals, plays a key role in conditions such as mood, eating disorders, and alcoholism. In plants, although serotonin has been suggested to be involved in several physiological roles, including flowering, morphogenesis, and adaptation to environmental changes, its regulation and functional roles are as yet not characterized at the molecular level. In this study, we found that serotonin is greatly accumulated in rice (Oryza sativa) leaves undergoing senescence induced by either nutrient deprivation or detachment, and its synthesis is closely coupled with transcriptional and enzymatic induction of the tryptophan biosynthetic genes as well as tryptophan decarboxylase (TDC). Transgenic rice plants that overexpressed TDC accumulated higher levels of serotonin than the wild type and showed delayed senescence of rice leaves. However, transgenic rice plants, in which expression of TDC was suppressed through an RNA interference (RNAi) system, produced less serotonin and senesced faster than the wild type, suggesting that serotonin is involved in attenuating leaf senescence. The senescence-retarding activity of serotonin is associated with its high antioxidant activity compared to either tryptophan or chlorogenic acid. Results of TDC overexpression and TDC RNAi plants suggest that TDC plays a rate-limiting role for serotonin accumulation, but the synthesis of serotonin depends on an absolute amount of tryptophan accumulation by the coordinate induction of the tryptophan biosynthetic genes. In addition, immunolocalization analysis revealed that serotonin was abundant in the vascular parenchyma cells, including companion cells and xylem-parenchyma cells, suggestive of its involvement in maintaining the cellular integrity of these cells for facilitating efficient nutrient recycling from senescing leaves to sink tissues during senescence.

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 Km and Vmax 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.

Melatonin promotes seminal root elongation and root growth in transgenic rice after germination

Abstract:  The effect of melatonin on root growth after germination was examined in transgenic rice seedlings expressing sheep serotonin N‐acetyltransferase (NAT). Enhanced melatonin levels were found in T3 homozygous seedlings because of the ectopic overexpression of sheep NAT, which is believed to be the rate‐limiting enzyme in melatonin biosynthesis in animals. Compared with wild‐type rice seeds, the transgenic rice seeds showed enhanced seminal root growth and an analogous number of adventitious roots 4 and 10 days after seeding on half‐strength Murashige and Skoog medium. The enhanced initial seminal root growth in the transgenic seedlings matched their increased root biomass well. We also found that treatment with 0.5 and 1 μm melatonin promoted seminal root growth of the wild type under continuous light. These results indicate that melatonin plays an important role in regulating both seminal root length and root growth after germination in monocotyledonous rice plants. This is the first report on the effects of melatonin on root growth in gain‐of‐function mutant plants that produce high levels of melatonin.

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

Abstract:  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.

Abstract:  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.

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 Km and Vmax values for SNAT at 55°C were 270 μm and 3.3 nmol/min/mg protein, whereas the Km and Vmax for ASMT were 222 μm and 9 nmol/min/mg protein, respectively. The catalytic efficiency (Vmax/Km) 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.

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 mmN‐acetyltryptamine during senescence process completely blocked melatonin synthesis.

Regulation of systemic energy homeostasis by serotonin in adipose tissues

Central serotonin (5-HT) is an anorexigenic neurotransmitter in the brain. However, accumulating evidence suggests peripheral 5-HT may affect organismal energy homeostasis. Here we show 5-HT regulates white and brown adipose tissue function. Pharmacological inhibition of 5-HT synthesis leads to inhibition of lipogenesis in epididymal white adipose tissue (WAT), induction of browning in inguinal WAT and activation of adaptive thermogenesis in brown adipose tissue (BAT). Mice with inducible Tph1 KO in adipose tissues exhibit a similar phenotype as mice in which 5-HT synthesis is inhibited pharmacologically, suggesting 5-HT has localized effects on adipose tissues. In addition, Htr3a KO mice exhibit increased energy expenditure and reduced weight gain when fed a high-fat diet. Treatment with an Htr2a antagonist reduces lipid accumulation in 3T3-L1 adipocytes. These data suggest important roles for adipocyte-derived 5-HT in controlling energy homeostasis.