Yeong Byeon

Chonnam National University, Gwangju, Gwangju, South Korea

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Publications (18)129.41 Total impact

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    ABSTRACT: Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae.
    Journal of Experimental Botany 09/2014; · 5.24 Impact Factor
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    ABSTRACT: Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in melatonin biosynthesis in both animals and plants. SNAT catalyzes serotonin into N-acetylserotonin, an immediate precursor for melatonin biosynthesis by N-acetylserotonin methyltransferase (ASMT). We cloned the SNAT gene from a gymnosperm loblolly pine (Pinus teada). The loblolly pine SNAT (PtSNAT) gene encodes 255 amino acids harboring a transit sequence with 67 amino acids and shows 67% amino acid identity with rice SNAT when comparing the mature polypeptide regions. Purified recombinant PtSNAT showed peak activity at 55°C with the Km (428 μm) and Vmax (3.9 nmol/mg protein) values. As predicted, PtSNAT localized to chloroplasts. The SNAT mRNA was constitutively expressed in all tissues, including leaf, bud, and pinecone, whereas the corresponding protein was detected only in leaf. In accordance with the exclusive SNAT protein expression in leaf, melatonin was detected only in leaf at 0.45 ng per gram fresh weight. Sequence and phylogenetic analysis indicated that the gymnosperm PtSNAT had high homology with SNATs from all plant phyla (even with cyanobacteria), and formed a clade separated from the angiosperm SNATs, suggestive of direct gene transfer from cyanobacteria via endosymbiosis.This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 09/2014; · 7.30 Impact Factor
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    ABSTRACT: 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 molecules in plant–pathogen interactions.This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 08/2014; · 7.30 Impact Factor
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    ABSTRACT: Although a plant N-acetylserotonin methyltransferase (ASMT) was recently cloned from rice, homologous genes appear to be absent in dicotyledonous plants. In order 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 O-methyltransferase (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 1,800 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.This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 07/2014; · 7.30 Impact Factor
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    ABSTRACT: 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 to seeds from transgenic plants expressing OaSNAT under the control of the constitutive maize ubiquitin promoter. This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 06/2014; · 7.30 Impact Factor
  • Yeong Byeon, Kyoungwhan Back
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    ABSTRACT: 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 week in the transgenic lines compared to 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. This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 02/2014; · 7.30 Impact Factor
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    ABSTRACT: A major goal of plant biotechnology is to improve the nutritional qualities of crop plants through metabolic engineering. Melatonin is a well-known bioactive molecule with an array of health-promoting properties, including potent antioxidant capability. To generate melatonin-rich rice plants, we first independently overexpressed three tryptophan decarboxylase isogenes in the rice genome. Melatonin levels were altered in the transgenic lines through overexpression of TDC1, TDC2, and TDC3; TDC3 transgenic seed (TDC3-1) had melatonin concentrations 31-fold higher than those of wild-type seeds. In TDC3 transgenic seedlings, however, only a doubling of melatonin content occurred over wild-type levels. Thus, a seed-specific accumulation of melatonin appears to occur in TDC3 transgenic lines. In addition to increased melatonin content, TDC3 transgenic lines also had enhanced levels of melatonin intermediates including 5-hydroxytryptophan, tryptamine, serotonin, and N-acetylserotonin. In contrast, expression levels of melatonin biosynthetic mRNA did not increase in TDC3 transgenic lines, indicating that increases in melatonin and its intermediates in these lines are attributable exclusively to overexpression of the TDC3 gene. This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 01/2014; · 7.30 Impact Factor
  • Yeong Byeon, Kyoungwhan Back
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    ABSTRACT: 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 to 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 since the effect of the relatively high temperature during the day was counteracted by the negative effect of the high light level. 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. This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 12/2013; · 7.30 Impact Factor
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    ABSTRACT: 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- and 4,054-fold higher at 55°C than at 30°C suggestive of increased melatonin production at high temperature in plants. This article is protected by copyright. All rights reserved.
    Journal of Pineal Research 10/2013; · 7.30 Impact Factor
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    ABSTRACT: N-acetylserotonin methyltransferase (ASMT) is the final enzyme in a biosynthetic pathway that produces melatonin. ASMT may play a rate-limiting role in the production of this bioactive molecule in plants. There are three ASMT genes (ASMT1-ASMT3) in the rice genome, but only ASMT1 has been functionally characterized. A major barrier to further progress in characterizing these genes has been a failure of functional expression within the Escherichia coli. Purified recombinant ASMT2 and ASMT3 are inactive in ASMT enzyme catalysis. To determine the biological functions of ASMT2 and ASMT3, we first overexpressed them in rice calli, which resulted in enhanced production of melatonin in the respective transgenic calli. To further corroborate the functions of ASMT2 and ASMT3 as ASMT genes, we generated stable transgenic rice plants. ASMT enzyme activity was increased in comparison with the wild type in T2 homozygous transgenic rice plants expressing three ASMT genes independently. When seedlings were treated with 1 mm N-acetylserotonin (NAS), leaf melatonin contents were higher in the three transgenic lines than in the wild type. There were no significant differences between the transgenic lines and the wild type without this treatment. ASMT1 and ASMT2 transcripts were highly expressed in stems and flowers, but ASMT3 was barely detectable in any of the plant organs. All three ASMT mRNAs were simultaneously induced in treatments with abscisic and methyl jasmonic acids.
    Journal of Pineal Research 08/2013; · 7.30 Impact Factor
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    ABSTRACT: 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.
    Journal of Pineal Research 08/2013; 55(1). · 7.30 Impact Factor
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    ABSTRACT: Serotonin N-acetyltransferase (SNAT) catalyzes conversion of serotonin into N-acetylserotonin, which is a direct precursor for melatonin biosynthesis in all organisms. Molecular cloning of plant SNAT from rice led to a screening for SNAT homolog genes in other species. We identified a cyanobacterium SNAT-like gene (cSNAT) that showed 56% amino acid homology with the rice SNAT. To confirm whether cSNAT encoded SNAT enzyme activity, we expressed cSNAT DNA in Escherichia coli and purified the cSNAT protein as a C-terminal His-tagged form. The purified cSNAT protein exhibited SNAT enzyme activities, transferring the acetyl group into either serotonin or tryptamine substrates. The optimum temperature was 55°C, but it was still highly active at 70°C, suggesting that cSNAT is a thermotolerant enzyme. The Km and Vmax were 823 μm and 1.6 nmol/min/mg protein, respectively. The cSNAT gene is highly conserved in all cyanobacterial taxa and seems to be an origin of SNAT in higher plants. The thermotolerance of cSNAT suggests that melatonin plays a role in the response to high-temperature stress. Further analysis of this role of melatonin in higher plants is needed.
    Journal of Pineal Research 07/2013; · 7.30 Impact Factor
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    ABSTRACT: Transgenic rice plants overexpressing a sheep serotonin N-acetyltransferase led to an enhanced production of melatonin with various physiological effects, including seminal root elongation and resistance against cold and oxidative stress, which raises the possibility that melatonin may alter gene expression profiles in the transgenic rice. Therefore, we performed a microarray analysis to investigate the regulatory role of melatonin using the melatonin-rich transgenic rice. We identified 260 and 204 genes that were up- or downregulated in the melatonin-rich transgenic rice when compared with the wild type. Of these, 20 upregulated genes were identified in the seedlings of melatonin-rich rice at more than twice the levels in the wild type (P < 0.05), while 23 downregulated genes were also detected. The representative upregulated genes included caleosin, a Ca(2+) -binding oil-body surface protein involved in the degradation of lipids stored in oil bodies and various signaling proteins such as a cyclin F-box protein and leucine-rich repeat protein. In contrast, jasmonate-induced protein, senescence-associated protein, and polygalacturonase were included in the downregulated gene group. These results suggest that melatonin has an important role in modulating a wide range of gene expression, reflecting its pleiotropic physiological roles in plant growth and development.
    Journal of Pineal Research 07/2013; · 7.30 Impact Factor
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    ABSTRACT: Rice tryptamine 5-hydroxylase (T5H) is the second enzyme in melatonin biosynthesis, catalyzing tryptamine into serotonin. Transgenic rice plants, in which the expression of endogenous T5H was either overexpressed or repressed, were examined for alteration in melatonin biosynthesis. Unexpectedly, the overexpression genotypes showed reduced levels of melatonin, while the repression genotypes had elevated levels with an average increase of fourfold. With regard to melatonin intermediates, tryptamine and serotonin levels decreased, but tryptophan and N-acetylserotonin were unaltered in the overexpression genotypes compared with the wild type. In contrast, the repression genotypes had sevenfold higher tryptamine levels than the wild type. In addition, tryptophan and 5-hydroxytryptophan were present at higher levels in the repression genotypes than in both the wild-type and the overexpression genotypes. The enhanced melatonin synthesis in the repression genotypes was closely associated with a transcriptional increase in TDC1. When these rice plants were challenged by oxidative stressors such as herbicides, much higher melatonin synthesis was also observed in the repression genotypes than in either the wild-type or overexpression genotypes. These results suggest that the tryptamine increase through the suppression of T5H plays an important signaling role in triggering melatonin biosynthesis in rice, although the exact role of tryptamine remains to be uncovered.
    Journal of Pineal Research 03/2013; · 7.30 Impact Factor
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    ABSTRACT: The regulation of reproduction-stage inducible melatonin biosynthesis in rice (Oryza sativa cv. Dongjin) was investigated. The flag leaf and panicle (flower) were collected from field-grown rice at three different reproductive stages: the preflowering stage, flowering stage, and postflowering stage. Melatonin synthesis was induced in the panicle, whereas no induction was observed in the flag leaf during the reproductive stages. The panicle displayed a peak melatonin level of 0.4 ng/g fresh weight (fw), which was six times that found in the flag leaf. The induction of melatonin biosynthesis was paralleled by the induction of corresponding genes and proteins such as tryptophan decarboxylase, tryptamine 5-hydroxylase, and N-acetylserotonin methyltransferase. In addition, melatonin induction was preceded by the accumulation of its precursor, tryptophan, in the panicle. These results suggest that the induction of melatonin during flower development is regulated by the transcriptional control of its biosynthesis genes and that melatonin may participate in flower development.
    Journal of Pineal Research 10/2012; · 7.30 Impact Factor
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    ABSTRACT: 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.
    Journal of Pineal Research 07/2012; · 7.30 Impact Factor
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    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.
    Journal of Pineal Research 06/2012; · 7.30 Impact Factor
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    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.
    Journal of Pineal Research 01/2012; 53(1):107-11. · 7.30 Impact Factor