Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

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.

Melatonin induction and its role in high light stress tolerance in Arabidopsis thaliana.

In plants, melatonin is a potent bioactive molecule involved in the response against various biotic and abiotic stresses. However, little is known of its defensive role against high light (HL) stress. In this study, we found that melatonin was transiently induced in response to HL stress in Arabidopsis thaliana with a simultaneous increase in the expression of melatonin biosynthetic genes, including serotonin N-acetyltransferase1 (SNAT1). Transient induction of melatonin was also observed in the flu mutant, a singlet oxygen (1 O2 )-producing mutant, upon light exposure, suggestive of melatonin induction by chloroplastidic 1 O2 against HL stress. An Arabidopsis snat1 mutant was devoid of melatonin induction upon HL stress, resulting in high susceptibility to HL stress. Exogenous melatonin treatment mitigated damage caused by HL stress in the snat1 mutant by reducing O2- production and increasing the expression of various ROS-responsive genes. In analogy, an Arabidopsis SNAT1-overexpressing line showed increased tolerance of HL stress concomitant with a reduction in malondialdehyde and ion leakage. A complementation line expressing an Arabidopsis SNAT1 genomic fragment in the snat1 mutant completely restored HL stress susceptibility in the snat1 mutant to levels comparable to that of wild-type Col-0 plants. The results of the analysis of several Arabidopsis genetic lines reveal for the first time at the genetic level that melatonin is involved in conferring HL stress tolerance in plants.

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.

Melatonin is required for H2 O2 - and NO-mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana.

Melatonin influences plant innate immunity through the mitogen-activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)-knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal-inducible1) kinases are responsible for triggering melatonin-induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H2 O2 and NO individually, but dependent on the combination of H2 O2 and NO. Moreover, melatonin-mediated induction of the expression of defense-related genes, such as PR1 and ICS1, was not altered in the H2 O2 -deficient rbohD/F-knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin-mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.

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.

Cadmium Disrupts Subcellular Organelles, Including Chloroplasts, Resulting in Melatonin Induction in Plants.

Cadmium is a well-known elicitor of melatonin synthesis in plants, including rice. However, the mechanisms by which cadmium induces melatonin induction remain elusive. To investigate whether cadmium influences physical integrities in subcellular organelles, we treated tobacco leaves with either CdCl2 or AlCl3 and monitored the structures of subcellular organelles-such as chloroplasts, mitochondria, and the endoplasmic reticulum (ER)-using confocal microscopic analysis. Unlike AlCl3 treatment, CdCl2 (0.5 mM) treatment significantly disrupted chloroplasts, mitochondria, and ER. In theory, the disruption of chloroplasts enabled chloroplast-expressed serotonin N-acetyltransferase (SNAT) to encounter serotonin in the cytoplasm, leading to the synthesis of N-acetylserotonin followed by melatonin synthesis. In fact, the disruption of chloroplasts by cadmium, not by aluminum, gave rise to a huge induction of melatonin in rice leaves, which suggests that cadmium-treated chloroplast disruption plays an important role in inducing melatonin in plants by removing physical barriers, such as chloroplast double membranes, allowing SNAT to gain access to the serotonin substrate enriched in the cytoplasm.

Mitogen-activated protein kinase pathways are required for melatonin-mediated defense responses in plants.

Melatonin enhances pathogen resistance by inducing the expression of a number of plant defense-related genes. To examine whether the melatonin-mediated pathogen resistance is associated with mitogen-activated protein kinase (MAPK) cascades, Arabidopsis and tobacco leaves were treated with melatonin and investigated for MAPK activation using an antiphospho-p44/42 MAPK (Erk1/2) monoclonal antibody. Two MAPKs, MPK3 and MPK6, were activated rapidly and transiently by 1 µm melatonin treatment in Arabidopsis. Its tobacco ortholog MAPKs were also activated. The activation of MPK3 and MPK6 by 2-hydroxymelatonin and N-acetylserotonin was also observed, albeit to a lesser degree than that by melatonin. Furthermore, MAPK activation by melatonin was uncoupled from G-protein signaling, because melatonin efficiently activated two MAPKs in a G-protein ß knockout mutant (agb1). Suppression of both MPK3 and MPK6 in transgenic Arabidopsis exhibited significant decreases in the induction of defense-related gene expression and pathogen resistance relative to wild-type plants. Using an array of MAP kinase kinase (MKK) knockout mutants, we found that four MKKs, namely MKK4, MKK5, MKK7, and MKK9, are responsible for the activation of MPK3 and MPK6 by melatonin, indicating that melatonin-mediated innate immunity is triggered by MAPK signaling through MKK4/5/7/9-MPK3/6 cascades.

Cloning and functional characterization of the Arabidopsis N-acetylserotonin O-methyltransferase responsible for melatonin synthesis.

The N-acetylserotonin O-methyltransferase (ASMT) gene encodes the enzyme that catalyzes the conversion of N-acetylserotonin to melatonin as the last step in melatonin biosynthesis. The first plant ASMT gene to be cloned was from rice. An orthologous gene encoding a protein with ASMT activity and only 39.7% amino acid sequence identity to the rice ASMT protein was recently isolated from apple (Malus zumi). The low homology of the apple ASMT sequence prompted us to screen the Arabidopsis genome for a homologous ASMT gene. The At4g35160 gene exhibited the highest sequence identity (31%) to the rice ASMT gene, followed by the At1g76790 gene with 29% sequence identity. We purified recombinant proteins expressed from the two Arabidopsis genes. The At4g35160 recombinant protein exhibited ASMT enzyme activity, but the At1g76790 recombinant protein did not; thus, we designated At4g35160 as an Arabidopsis thaliana ASMT (AtASMT) gene. The AtASMT protein catalyzed the conversion of N-acetylserotonin to melatonin and serotonin to 5-methoxytryptamine with Vmax values of 0.11 and 0.29 pkat/mg protein, respectively. However, AtASMT exhibited no caffeic acid O-methyltransferase activity, suggesting that its function was highly specific to melatonin synthesis. AtASMT transcripts were induced by cadmium treatment in Arabidopsis followed by increased melatonin synthesis. Similar to other ASMT proteins, AtASMT was localized in the cytoplasm and its ectopic overexpression in rice resulted in increased ASMT enzyme activity and melatonin production, indicating the involvement of AtASMT in melatonin synthesis.

Cloning and characterization of the serotonin N-acetyltransferase-2 gene (SNAT2) in rice (Oryza sativa).

The penultimate enzyme in melatonin synthesis is serotonin N-acetyltransferase (SNAT), which exists as a single copy in mammals and plants. Our recent studies of the Arabidopsis snat-knockout mutant and SNAT RNAi rice (Oryza sativa) plants predicted the presence of at least one other SNAT isogene in plants; that is, the snat-knockout mutant of Arabidopsis and the SNAT RNAi rice plants still produced melatonin, even in the absence or the suppression of SNAT expression. Here, we report a molecular cloning of an SNAT isogene (OsSNAT2) from rice. The mature amino acid sequences of SNAT proteins indicated that OsSNAT2 and OsSNAT1 proteins had 39% identity values and 60% similarity. The Km and Vmax values of the purified recombinant OsSNAT2 were 371 µm and 4700 pmol/min/mg protein, respectively; the enzyme's optimal activity temperature was 45°C. Confocal microscopy showed that the OsSNAT2 protein was localized to both the cytoplasm and chloroplasts. The in vitro enzyme activity of OsSNAT2 was severely inhibited by melatonin, but the activities of sheep SNAT (OaSNAT) and rice OsSNAT1 proteins were not. The enzyme activity of OsSNAT2 was threefold higher than that of OsSNAT1, but 232-fold lower than that of OaSNAT. The OsSNAT1 and OsSNAT2 transcripts were similarly suppressed in rice leaves during the melatonin induction after cadmium treatment. Phylogenetic analyses indicated that OsSNAT1 and OsSNAT2 are distantly related, suggesting that they evolved independently from Cyanobacteria prior to the endosymbiosis event.

Melatonin biosynthesis requires N-acetylserotonin methyltransferase activity of caffeic acid O-methyltransferase in rice.

Caffeic acid O-methyltransferase (COMT) methylates N-acetylserotonin into melatonin; that is, it has N-acetylserotonin O-methyltransferase (ASMT) activity. The ASMT activity of COMT was first detected in Arabidopsis thaliana COMT (AtCOMT). To confirm the involvement of COMT on melatonin synthesis in other plant species, the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT) was evaluated. Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The K m and V max values for the ASMT activity of OsCOMT were 243 µM and 2400 pmol min(-1) mg protein(-1), which were similar to those of AtCOMT. Similar to AtCOMT, OsCOMT was localized in the cytoplasm. In vitro ASMT activity was significantly inhibited by either caffeic acid or quercetin in a dose-dependent manner. Analogously, in vivo production of melatonin was significantly inhibited by quercetin in 4-week-old detached rice leaves. Lastly, the transgenic rice plants overexpressing rice COMT showed an increase in melatonin levels whereas transgenic rice plants suppressing the rice COMT had a significant decrease on melatonin levels, suggestive of the direct role of COMT in melatonin biosynthesis 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.

Arabidopsis serotonin N-acetyltransferase knockout mutant plants exhibit decreased melatonin and salicylic acid levels resulting in susceptibility to an avirulent pathogen.

Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis.

Coordinated regulation of melatonin synthesis and degradation genes in rice leaves in response to cadmium treatment.

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.

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress.

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe(2+) into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H2O2 contents as well as sustained Fv/Fm compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.

Arabidopsis RTNLB1 and RTNLB2 Reticulon-like proteins regulate intracellular trafficking and activity of the FLS2 immune receptor.

Receptors localized at the plasma membrane are critical for the recognition of pathogens. The molecular determinants that regulate receptor transport to the plasma membrane are poorly understood. In a screen for proteins that interact with the FLAGELIN-SENSITIVE2 (FLS2) receptor using Arabidopsis thaliana protein microarrays, we identified the reticulon-like protein RTNLB1. We showed that FLS2 interacts in vivo with both RTNLB1 and its homolog RTNLB2 and that a Ser-rich region in the N-terminal tail of RTNLB1 is critical for the interaction with FLS2. Transgenic plants that lack RTNLB1 and RTNLB2 (rtnlb1 rtnlb2) or overexpress RTNLB1 (RTNLB1ox) exhibit reduced activation of FLS2-dependent signaling and increased susceptibility to pathogens. In both rtnlb1 rtnlb2 and RTNLB1ox, FLS2 accumulation at the plasma membrane was significantly affected compared with the wild type. Transient overexpression of RTNLB1 led to FLS2 retention in the endoplasmic reticulum (ER) and affected FLS2 glycosylation but not FLS2 stability. Removal of the critical N-terminal Ser-rich region or either of the two Tyr-dependent sorting motifs from RTNLB1 causes partial reversion of the negative effects of excess RTNLB1 on FLS2 transport out of the ER and accumulation at the membrane. The results are consistent with a model whereby RTNLB1 and RTNLB2 regulate the transport of newly synthesized FLS2 to the plasma membrane.

Melatonin as a signal molecule triggering defense responses against pathogen attack in Arabidopsis and tobacco.

Melatonin plays pleiotropic roles in both animals and plants. The possible role of melatonin in plant innate immune responses was recently discovered. As an initial study, we employed Arabidopsis to determine whether melatonin is involved in defense against the virulent bacterial pathogen Pseudomonas syringae DC3000. The application of a 10 µM concentration of melatonin on Arabidopsis and tobacco leaves induced various pathogenesis-related (PR) genes, as well as a series of defense genes activated by salicylic acid (SA) and ethylene (ET), two key factors involved in plant defense response, compared to mock-treated leaves. The induction of these defense-related genes in melatonin-treated Arabidopsis matched an increase in resistance against the bacterium by suppressing its multiplication about ten-fold relative to the mock-treated Arabidopsis. Like melatonin, N-acetylserotonin also plays a role in inducing a series of defense genes, although serotonin does not. Furthermore, melatonin-induced PR genes were almost completely or partially suppressed in the npr1, ein2, and mpk6 Arabidopsis mutants, indicative of SA and ET dependency in melatonin-induced plant defense signaling. This suggests that melatonin may be a novel defense signaling molecule in plant-pathogen interactions.

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.

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 Km and Vmax values for ASMT activity were 233 µm and 1800 pmol/min/mg protein, while the Km and Vmax values for COMT activity were 103 µm and 564,000 pmol/min/mg protein, respectively. The catalytic efficiency (Vmax /Km ) 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.

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.