유전자변형 작물이 절지동물에 미칠 수 있는 잠재적인 부정적 영향은 유전자변형 작물의 주요한 환경위해성의 하나로 여겨지고 있다. 본 연구에서는 PPO (protoporphyrinogen oxidase) 저해 제초제 내성 유전자변형 벼가 절지동물에 미칠 수 있는 영향을 평가하기 위하여 절지동 물의 다양성과 군집구조를 조사하였다. 절지동물은 야외포장에서 벼의 생육기간 동안 황색점착트랩을 이용하여 채집하였다. 유전자변형 벼는 채 집된 절지동물군집의 다양도 지수에 유의한 영향을 주지 않았다. 또한 다변량분석(PerMANOVA, NMDS) 결과에서도 절지동물군집 구조는 채집시기에 따라 달랐지만 벼의 유전형(유전자변형 또는 비변형)에 의해 영향을 받지 않았다.

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 ASMT activity of COMT in other plant species, we evaluated the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT). Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The Km and Vmax values for the ASMT activity of OsCOMT were 243 μm and 2,400 pmol/min/mg protein, 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, suggestive of a positive role of COMT in melatonin biosynthesis in plants.

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.

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.

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

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.

Rice (Oryza sativa) is the most important staple food of over half the world’s population. This study was conducted to evaluate the possible impact of transgenic rice cultivation on the soil microbial community. Microorganisms were isolated from the rhizosphere of GM and non-GM rice cultivation soils. Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with GM and non-GM rice were similar to each other, and there was no significant difference between GM and non-GM rice. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM rice were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in GM and non-GM rice cultivated soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed similar patterns, but didn’t show significant difference to each other. DNAs were isolated from soils cultivating GM and non-GM rice and analyzed for persistence of inserted gene in the soil by using PCR. The PCR analysis revealed that there were no amplified protox gene in soil DNA. These data suggest that transgenic rice does not have a significant impact on soil microbial communities, although continued research may be necessary.

There are many evidences that carotenoids may act as antioxidants and protect humans from serious disorders such as skin degeneration and aging, cardiovascular disease, certain types of cancer, and age-related diseases of the eye. Carrots (Daucus carota L.) are consumed as an important dietary source of b-carotene, a-carotene and lutein. Astaxanthin, a keto-carotenoid has been used to raise red color of fish body and to improve immune activity in fish-breeding industry. In this study, transgenic carrot plants were generated to overproduce carotenoids including astaxanthin, a non-natural ketocarotenoid in this plant, using an efficient storage root-expression system. Among the nineteen transgenic carrot plants, transformed by a storage root-specific (ibMads) or a storage root (ibAGP1) or the constitutive CaMV35S promoters with three genes involved in carotenoid synthesis [Psy (Phytoen synthase), Crtl (Lycopen-β-cyclase), CrtO (β-carotene ketolase)], transgenic plants with ibAGP1 promoter, an amyloplast targeting sequence (TP1) and a single CrtO gene gave high content of keto-carotenoids and b-carotene. For fish body coloration, carotenoid extract or astaxanthin significantly made the body color of red seabreams more reddish than those of normal diet-fish in the 3 weeks feeding. In addition, the serum lysozyme activity in carotene-treated fish was significantly higher than that in normal diet-fed fish (P<0.05) in the 6 weeks feeding. In these cases, neither carotenoid extract- nor astaxanthin-contained diet did influence on growth rate and food utilization in red seabreams. These results suggested that carotenoid extract prepared in the present study may be useful in the body coloration and the enhancement of nonspecific immune response of red seabreams. Meanwhile, b-carotene (50 mM) up-regulated peroxisome proliferator-activated receptor a expression (PPAR-a) by about two fold in CV-1 cells, while the carotenoid extracts and astaxanthin failed to affect on the expression. Carotenoid extracts (250 mg/ml) from wild type carrot or transgenic carrots showed moderate DPPH scavenging activity.

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway in the expressed cytoplasm or the plastids, were compared with non-trangenic rice plants in their growth characteristics such as tiller number, plant height, biomass, and yield. Transgenic rice plants of ~textrmT3 generation had 8 to 15 % and 25 to 43% increases in tiller number compared to non-transgenic rice plants at 4 and 8 weeks after transplanting(WAT); similar values were observed for ~textrmT4 generation at 4 and 8 WAT. However, the plant height in both ~textrmT3 and ~textrmT4 generations was similar between transgenic rice plants and non-transgenic rice plants at 4 and 8 WAT. Transgenic rice plants had 13 to 32% increase in above-ground biomass and 9 to 28% increase in grain yield compared to non-transgenic rice plants, demonstrating that biomass and yield correlate with each other. The increased grain yield of the transgenic rice plants was closely associated with the increased panicle number per plant. The percent of filled grain, thousand grains and spikelet number per panicle were similar between transgenic and non-transgenic rice plants. Generally, the growth and yield of transgenic generations (~textrmT2 , ~textrmT3 , and ~textrmT4 ) and gene expressing sites (cytoplasm-expressed and plastid-targeted transgenic rice plants) were similar, although they slightly varied with generations as well as with gene expressing sites. The transgenic rice plants had promotive effects, indicating that regulation of the porphyrin pathway by expression of B. subtilis Protox in rice influences plant growth and yield.