Yeong Byeon

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.

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.

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 synthesis in rice seedlings in vivo is enhanced at high temperatures and under dark conditions due to increased serotonin N‐acetyltransferase and N‐acetylserotonin methyltransferase activities

Temperature and light are important environmental factors for plant growth and development. The final two enzymes in the melatonin synthesis pathway in plants are serotonin N‐acetyltransferase (SNAT) and N‐acetylserotonin methyltransferase (ASMT), which have thermophilic characteristics. Thus, the effects of temperature and light on melatonin synthesis in rice seedlings were investigated. Here, we demonstrated that melatonin levels increased as temperature increased when rice seedlings were exposed to various temperatures for 1 hr. Moreover, the relative melatonin levels were higher in the dark. For example, exposure of rice seedlings to 1‐hr darkness at 55°C resulted in a melatonin yield of 4.9 ng/g fresh weight (fw), compared with 2.95 ng/g fw under light conditions. Temperature‐dependent melatonin synthesis was closely associated with an increase in both SNAT and ASMT activities, but not with transcript levels of melatonin biosynthetic genes. The daily melatonin levels in field‐grown rice plants were unaffected as the positive effect of the relatively high temperature during the day was counteracted by the negative effect of the high light. The opposite effect occurred during the night, in which the positive effect of darkness on melatonin synthesis was counteracted by the negative effect of a low temperature.

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

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

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.

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.

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.

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.

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.