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Hyoungyool Lee,Kyoungwhan Back 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
Melatonin plays pleiotropic roles in both animals and plants. Among them, the possible role of melatonin in the innate immune response in plants was emerging recently. As an initial study, we employed Arabidopsis to see whether melatonin is involved in the defense system against a virulent bacterial pathogen Psudomonas syringae DC3000. It was obviously observed that melatonin application of 10 μm concentration onto 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 the plant defense response compared to the mock-treated Arabidopsis and tobacco leaves, respectively. The induction of these defense-related genes in the melatonin treated Arabidopsis was well matched with an increase in resistance against pathogenic bacterium by suppressing its multiplication with about 10 fold relative over the mock-treated Arabidopsis. Furthermore, melatonin induced PR genes were almost completely or partially suppressed in npr1, ein2, and mpk6 Arabidopsis mutants indicative of SA and ET dependency of melatonin in plant defense signaling. These results suggest that melatonin may play a novel defense signaling molecule in plant-pathogen interaction
Presence of melatonin 2-hydroxylase in rice (Oryza sativa) plants
Yeong Byeon,Kyoungwhan Back 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
Although melatonin biosynthetic genes from plants have been cloned, the melatonin catabolism mechanisms remain unclear. To clone the genes responsible for melatonin metabolism, we ectopically expressed 35 fulllength cDNAs of rice 2-oxoglutarate-dependent dioxygenase (2-ODD) in Escherichia coli and purified the corresponding recombinant proteins. In vitro 2-ODD assays showed four independent 2-ODD proteins that were able to catalyze melatonin into 2-hydroxymelatonin, exhibiting melatonin 2-hydroxylase (M2H). These M2H proteins had peak activities at pH 8.0 and 30°C. The Km ranged from 121 μM to 371 μM with the Vmax ranging from 1.7 to 18.5 pkat/mg protein, respectively. The M2H enzyme activities were dependent on cofactors such as α-ketoglutarate, ascorbate, and Fe2+, similar to the 2-ODD enzymes. M2H activity was inhibited by prohexadione-Ca, an inhibitor of 2-ODD, in a dose-dependent manner. M2H activity was high in the roots of rice seedlings, concurrent with high transcription levels of 2-ODD 21, suggesting that 2-ODD 21 was a major gene for M2H activity. Analogous to the high M2H activity in the roots, 2-hydroxymelatonin was found in large quantities in roots treated with melatonin. These results suggest that melatonin was metabolized into 2-hydroxymelatonin by the M2H genes in plants, but the physiological significance of 2-hydroxymelatonin remains to be examined in the future.
Study of transgenic rice plants in rich expressed sheep serotonin N-Acetyltransferase
Yeong Byeon,Hyoung Yool Lee,Kyoungwhan Back 한국육종학회 2015 한국육종학회 심포지엄 Vol.2015 No.07
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 cytoplasmexpressed 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.
Yeong Byeon,Nansook Lee,Kyungjin Lee,Kyoungwhan Back 한국육종학회 2014 한국육종학회 심포지엄 Vol.2014 No.07
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.
Yang, Kiwoung,Jung, Sunyo,Lee, Yonghyuk,Back, Kyoungwhan Academic Press 2006 Pesticide biochemistry and physiology Vol.86 No.3
<P><B>Abstract</B></P><P>Protoporphyrinogen oxidase (Protox) of <I>Myxococcus xanthus</I> (<I>Mx</I> Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, <I>Mx</I> Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the <I>Mx</I> Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the <I>Arabidopsis</I> Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified <I>Mx</I> Protox gene was used to generate transgenic rice plants <I>via Agrobacterium</I>-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content <I>Mx</I> Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native <I>Mx</I> Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.</P>