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Plant-specific NAC (NAM, ATAF, and CUC)- domain proteins play important roles in plant development and stress responses. This study presents a comprehensive overview of GmSNAC genes. Eight of 71 GmSNAC genes differentially induced by abiotic stresses were identified. Based on the structural characteristics and phylogenetic relationships of Arabidopsis, rice, and soybean NACs, the GmSNACs were divided into five subgroups. Five GmSNAC proteins were localized in the nucleus and three GmSNAC proteins containing transmembrane domains were found in the nucleus and in other fractions. Based on the transcriptional activities of eight GmSNAC proteins fused to the GAL4 DB, it seems that the respective GmSNAC might play a transcriptional activator or repressor role in yeast. Here, we identify the roles of GmSNAC49 in stress responses and other processes. The overexpression of GmSNAC49 in Arabidopsis produced increased tolerance to drought stress compared to WT plants. Real-time (q)RT-PCR analyses suggest that overexpression of GmSNAC49 resulted in the upregulation of genes involved in the drought and/or ABA signaling pathway. These results suggest that GmSNAC49 might play a positive role in drought stress tolerance by regulating drought-responsive genes in Arabidopsis. Overall, the results of this systematic analysis of the GmSNAC family responsive to abiotic stress will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars.

Sasa coreana Nakai (SCN) is a medicinal plant commonly used against inflammation. However, the underlined mechanisms against skin inflammation is poorly understood. The present study investigated the effects of SCN leave extract on ear inflammation. To this aim, six-week-old male ICR mice was subjected to 12-O-tetradecanoyl-phorbol-13-acetate induce ear edema, which were then topically treated with the leave extract of SCN. Ear thickness, weight, and morphological changes were recorded to ensure the induction of ear edema. Further, histological analysis and protein expression for inflammatory markers were also recorded to validate the study. Topical treatment with SCN repressed TPA-induced ear edema in a dose-dependent manner. Further, SCN treatment significantly antagonized the protein expression of MAP kinase signaling pathway and reduced the effect of TPA-induced NF-κB activation, sequentially, deactivated its transcriptional targets in a dose-dependent manner. Collectively, the study suggested that SCN could be a useful therapeutic agent against skin inflammation.

Soybean [Glycine max (L.) Merr.] is an important crop for vegetable oil production, and is a major protein source worldwide. Because of its importance as a crop, genetic transformation has been used extensively to improve its valuable traits. Soybean mosaic virus (SMV) is one of the most well-known viral diseases affecting soybean. Transgenic soybean plants with improved resistance to SMV were produced by introducing HC-Pro coding sequences within RNA interference (RNAi) inducing hairpin construct via Agrobacterium-mediated transformation. During an experiment to confirm the response of transgenic plants (T2) to SMV infection, no T2 plants from lines #2 (31/31), #5 (35/35) or #6 (37/37) exhibited any SMV symptoms, indicating strong viral resistance (R), whereas NT (non-transgenic wild type) plants and those from lines #1, #3 and #4 exhibited mild mosaic (mM) or mosaic (M) symptoms. The northern blot analysis showed that three resistant lines (#2, #5 and #6) did not show the detection of viral RNA accumulation while NT, EV (transformed with empty vector carrying only Bar) and lines #1, #3 and #4 plants were detected. T3 seeds from SMV-inoculated T2 plants were harvested and checked for changes in seed morphology due to viral infection. T3 seeds of lines #2, #5 and #6 were clear and seed coat mottling was not present, which is indicative of SMV resistance. RT-PCR and quantitative real-time PCR showed that T3 seeds from the SMV-resistant lines #2, #5 and #6 did not exhibit any detection of viral RNA accumulation (HC-Pro, CP and CI), while the viral RNA accumulation was detected in SMV-susceptible lines #1, #3 and #4 plants. During the greenhouse test for viral resistance and yield components, T3 plants from the SMV-inoculated transgenic lines #2, #5 and #6 showed viral resistance (R) and exhibited a more favorable average plant height, number of nodes per plant, number of branches per plant, number of pods per plant and total seed weight with statistical significance during strong artificial SMV infection than did other plant lines. In particular, the SMV-resistant line #2 exhibited superior average plant height, pod number and total seed weight with highly significance. According to our results, RNAi induced by the hairpin construct of the SMV HC-Pro sequence effectively confers much stronger viral resistance than did the methods used during previous trials, and has the potential to increase yields significantly. Because of its efficiency, the induction of RNAi-mediated resistance will likely be used more frequently as part of the genetic engineering of plants for crop improvement.

We used the next-generation Illumina/Solexa HiSeq2000 platform on RNA analysis to investigate the transcriptome of Astragalus membranaceus hairy roots in response to 100 μM methyl jasmonate (MeJA). In total, 77,758,230 clean reads were assembled into 48,636 transcripts (average length of 1398 bp), which were clustered into 23,658 loci (genes). Of these, 19,940 genes were annotated by BLASTx searches. In addition, DESeq analysis showed that 2127 genes were up-regulated, while 1247 genes were down-regulated by MeJA. Seventeen novel astragaloside (AST) biosynthetic genes and seven novel calycosin and calycosin-7-O-β-D-glucoside (CG) biosynthetic genes were isolated. The accumulation of ASTs, calycosin, and CG increased significantly in MeJA-treated hairy roots compared with control hairy roots. Our findings will provide a valuable resource for molecular characterization of AST, calycosin, and CG biosynthetic pathways and may lead to new approaches to maximize their production and biomass productivity in the hairy roots of A. membranaceus.

Abiotic stresses are the major concern in recent years as their effect on world food production is constantly increasing. We have obtained salt tolerant Arabidopsis lines overexpressing SaRBP1 (Suaeda asparagoides RNA binding protein 1) of a Korean halophyte, S. asparagoides. Homozygous T3 Arabidopsis transgenic lines were developed and used for salt stress tolerance studies. The transgenic seedlings displayed tolerance to salt and mannitol compared to the wild type (WT) seedlings. Transgenic lines produced longer primary roots, more fresh weight, and higher number of lateral roots than WT. In planta stress tolerance assay results showed that the survival rates of transgenic plants were significantly higher than WT plants. Transgenic lines showed delayed germination under 200 mM NaCl stress. In addition, the transgenics showed higher water retention ability than WT. Subcellular localization results revealed that SaRBP1 was targeted to the cytoplasm. Northwestern blot analysis results confirmed the RNA binding property of SaRBP1. Quantitative Real-Time Polymerase Chain Reaction results revealed that many stress marker genes were upregulated by SaRBP1 overexpression. Thus, our data demonstrate that SaRBP1 overexpression lines are tolerant to salt stress. Hence, this is the first report for the functional characterization of SaRBP1, a novel RBP gene isolated from S. asparagoides cDNA library.

Background Epidermal growth factor receptor (EGFR) inhibitors are a standard treatment for various malignant tumors, especially in colon cancer and lung cancer. The most common adverse reaction of EGFR inhibitors is skin toxicity, including acneiform rash, xerosis, paronychial inflammation, pruritus, photosensitivity, and hair/eyelash alterations. Even though EGFR inhibitor-related skin adverse events (ERSEs) are predictive marker of EGFR inhibitors treatment survivals, those are frequently also the reason for premature termination of anti-EGFR therapy. It is therefore important to understand the mechanisms underlying the skin toxicities caused by EGFR inhibition to improve anti-EGFR–based cancer therapies and minimize debilitating side effects for patients. Methods Twelve patients’ ERSEs skin biopsies which were treated with EGFR inhibitors were compared to same numbers of controlled skin biopsies those who did not use EGFR inhibitors. We evaluated Ki-67, EGFR, Melan-A, interleukin-17 (IL-17), and tumor necrosis factor-α (TNF-α) expression based on immunohistochemical (IHC) stains between 2 groups. Among them, five patients were treated with EGF ointment for ERSE. We also observed skin changes before and after EGF ointment. Results Control and ERSE group’s Ki-67 expression of epidermis were 40.8% vs 21.2% (P = 0.015). EGFR presentation range of epidermis was 98.3% vs 84.6% in control and ERSEs group, respectively (P = 0.001). We could observe 14.2% vs 8.1% (P = 0.069) of Melan-A IHC stain. In the IL-17, ERSE group’s IL-17 expression intensity (16.1) was higher than control group (9.8) (P = 0.038). Much higher TNF-α expression intensity (13.3) was also observed in ERSE group compared control group (7.9) (P = 0.037). After treatment with EGF ointment of 5 patients, values of Ki-67, EGFR, Melan-A, IL-17, and TNF-α were changed to 28%, 94%, 8.2%, 12.5 and 10, respectively. Conclusions Treatment with EGFR inhibitors decreased expression of Ki-67 and EGFR in the patients’ dermis. This seems to lead to secondary inflammation of the skin. The treatment of EGF ointment for ERSEs is thought to normalize EGFR level and the inflammatory response to some extent. Clinical trial identification NCT02284139. Legal entity responsible for the study The authors. Funding Daewoong Pharmaceutical Company. Disclosure J.M. Kim: Full / Part-time employment: Daewoong pharmacy. J.E. Choo: Full / Part-time employment: Daewoong pharmacy. All other authors have declared no conflicts of interest.

The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

Heat stress severely affects plant growth and development causing crop loss worldwide. Classical type I DnaJ proteins (also called as J-proteins, J-domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. In this study, we have cloned and characterized a novel gene GmDjp1 (G lycine m ax DnaJ protein 1) encoding a type III J-protein of which function has not been identified in plant. Deduced amino acid sequences of GmDjp1 show the highest homology with a J-protein from Medicago truncatula legume plant (83 %) and with Arabidopsis thaliana type III J-class proteins, atDjC53 (77 %) and atDjC32 (50 %). DNA blot analysis revealed that GmDjp1 exists as a 2-copy gene in soybean genome. GmDjp1 mRNA was induced by a broad spectrum of abiotic stresses, including wounding, heat-shock, dehydration, cold or high-salinity stress, suggesting its role in the signaling events in the abiotic stress-related defense response. Subcellular localization studies demonstrated that the GmDjp1-GFP fusion protein was localized in the nucleus. Differential RNA expression of GmDjp1 by heat-shock stress inspired us to test heat-shock tolerance of GmDjp1in E. coli. Heterologous expression of GmDjp1 conferred tolerance to high temperature stress in E. coli. This report provides strong evidence that GmDjp1 may play a critical role during heat-shock stress in cell.

Two cDNA clones encoding RNA binding proteins (RBPs) were isolated from a cDNA library constructed from salt-treated leaf tissues of wild radish (Raphanus sativus var. hortensis for raphanistroide). The deduced amino acid sequence of either RsRBP1 or RsGRP1 contains an RNA-recognition motif (RRM) at the carboxy or amino terminal. Comparative sequence analysis of RsRBP1 reveals extensive homology (63–84%) to known RBPs from other plants. RsGRP1 was shown to be most homologous to AtGRP7 (93%) out of eight members of Arabidopsis glycine-rich RBPs. Transcript levels of RsRBP1 was up-regulated slowly and reached its maximum at 9 h during salt stress. On the other hand, RNA expression of RsGRP1 was up-regulated rapidly but significantly was reduced at 9 h after salt stress. The RsRBP1 and RsGRP1 proteins were detected in the nucleus and cytoplasm. Characterization of the transgenic Arabidopsis plants overexpressing RsRBP1 and RsGRP1 revealed that both transgenic lines displayed enhanced growth under the osmotic stress conditions. Overexpression of RsGRP1 resulted in delayed germination rates under the osmotic stress conditions, whereas RsRBP1 overexpression Arabidopsis did not display any difference in germination rates during osmotic stress. These results suggest that RsRBP1 and RsGRP1 may be involved in the responses to osmotic stress in plant.

Drought and high salinity stresses often imposes adverse effects on crop yield. MYB transcription factors have been shown to be an important regulator in defense responses to these environmental stresses. In this study, we have cloned and characterized a soybean gene GmMYB184 ( Glycine max MYB transcription factor 184). Deduced amino acid sequences of GmMYB184 show highest homology with that from Vitis vinifera legume plant (75%). Different ex-pression patterns of GmMYB184 mRNA were observed subjected to drought, cold, high salinity stress and abscisic acid treatment, suggesting its role in the signaling events in the osmotic stress-related defense response. Subcellular localization studies demonstrated that the GFP- GmMYB184 fusion protein was localized in the nucleus. Using the yeast assay system, the C-terminal region of GmMYB184 was found to be essential for the transactivation activity. These results indicate that the GmMYB184 may play a role in abiotic stress tolerance in plant.

Recent studies have shown that an intron is not merely “junk”, but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5′- and 3′-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, AYGW GTABABGH and TVNC/TAGGHTV for the 5′SS and 3′SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3′SS region was relatively more conserved than the 5′SS-BP region. We speculate that length of the BP-3′SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.

To examine the neuroprotective effects of Glycine max, we tested its protection against the glutamate-induced toxicity in primary cortical cultured neurons. In order to clarify the neuroprotective mechanism(s) of this observed effect, isolation was performed to seek and identify active fractions and components. From such fractionation, two triterpene glycosides, 3-O-[alpha-L-rhamnopyranosyl(1-2)-beta-D-glucopyranosyl(1-2)-beta-D-glucuronopyranosyl]olean-12-en-3 beta,22 beta,24-triol (1) and 3-O-[beta-D-glucopyranosyl(1-2)-beta-D-galactopyranosyl(1-2)-beta-D-glucuronopyranosyl]olean-12-en-3 beta,22 beta,24-triol (2) were isolated with the methanol extracts with of air-dried Glycine max. Among these compounds, compound 2 exhibited significant neuroprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50% at concentrations ranging from 0.1 mu M to 10 mu M. Therefore, the neuroprotective effect of Glycine max might be due to the inhibition of glutamate-induced toxicity by triterpene glycosides.

Apoptosis, the main type of programmed cell death, plays an essential role in a variety of biological events. Whereas "classical" apoptosis is dependent on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. To develop new anticancer agents, oleifolioside A was isolated from Dendropanax morbifera Leveille and the biochemical mechanisms of oleifolioside A-induced apoptosis in HeLa cells were investigated. Exposure to oleifolioside A resulted in caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Oleifolioside A treatment induced up-regulation of Bad, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, apoptosis-inducing factor (AIF), endonuclease G (EndoG), and apoptosis induction. This is the first report of anticancer activity of oleifolioside A, and nuclear translocation of AIF and EndoG in oleifolioside A-treated HeLa cells might represent an alternative death signaling pathway in the absence of caspase activity.

The expression of matrix metalloproteinase (MMPs)-9 is critical for cell migration and can lead to invasion and metastasis of cancer cells. In the present study, we examined the inhibitory effects of JNP3, a new compound which was isolated from traditional Chinese medicine, on cell invasion and MMP-9 activation in phorbol myristate acetate (PMA)-induced MCF-7 cells. Treatment with JNP3 significantly and selectively inhibited PMA-induced MMP-9 secretion, mRNA expression and protein levels, and these results led to reduction of cell invasion and migration in PMA-induced MCF-7 cells. The results of MMP-9 promoter assay and EMSA showed that JNP3 specifically inhibited PMA-induced MMP-9 gene expression by blocking NF-κB-dependent transcriptional activity. In addition, PMA-induced phosphorylation of ERK1/2 and JNK were suppressed by JNP3 treatment, whereas the phosphorylation of p38 MAPK was not affected by JNP3. These results suggest that JNP3 can be potential anti-cancer agents through specific inhibition of NF-κB-dependent MMP-9 gene expression.

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system.

The present study evaluated the anticomplement effects from isolated compounds of black bean in classical pathway complement system. Using column chromatograph, four compounds kaempferol 3-O-β-D-glucopyranoside (1), kaempferol 5-O-β-D-glucopyranoside (2), 3-O-[α-L-rhamnopyranosyl(1-2)-β-D-glucopyranosyl(1-2)-β-D-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (3) and 3-O-[β-D-glucopyranosyl(1-2)-β-D-galactopyranosyl(1-2)-β-D-glucuronopyranosyl]olean-12-en-3β,22β,24-triol (4) were isolated and evaluated for in vitro anticomplement activity. 3 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 98.2 μg/ml. This is the first report of anticomplement activity of isolated compounds from black bean.

A mungbean low temperature-inducible VrPrx1 encoding 2-Cys peroxiredoxin (2-Cys Prx) was cloned by subtractive suppression hybridization. The deduced VrPrx1 amino acid sequence showed highest sequence homology to 2-Cys Prxs of Phaseolus vulgaris (95%), Pisum sativum (89%), and Arabidopsis thaliana (87%). VrPrx1 RNA and protein levels were increased by low temperature, hydrogen peroxide (H2O2), and wounding but decreased by high salinity, drought, and exogenous abscisic acid. Recombinant His-tagged VrPrx1 recombinant protein protected DNA and glutamine synthetase activity from degradation via the thiol/Fe(III) oxygen mixed-function oxidation system, and exhibited peroxidase activity to H2O2 in the presence of the reducing agent dithiothreitol (DTT) in vitro. The oxidized dimers and oligomers of the VrPrx1 recombinant protein were reduced to monomers by DTT or thioredoxin. Subcellular localization studies confirmed that VrPrx1-GFP was targeted to the plastid. To evaluate the function of VrPrx1 in planta, the antioxidant activities and photosynthetic efficiency were investigated in VrPrx1-overexpressing Arabidopsis plants. VrPrx1 ectopic expression conferred improved photosynthetic efficiency under oxidative stress conditions. Hence, mungbean VrPrx1 may play an important role in protecting the photosynthetic apparatus against oxidative and abiotic stress conditions.

The present study evaluated the anticomplement effects from isolated compounds of Sorghum bicolor in classical pathway complement system. Using column chromatograph, three compounds; Sorgoleone-362 (1), Sorgoleone-360 (2) and Sorgoleone-386 (3) were isolated and evaluated for in vitro anticomplement activity. Sorgoleone-386 showed inhibitory activity against complement system with 50% inhibitory concentrations (IC(50)) values of 148.3μg/ml. This is the first report of anticomplement activity of isolated compounds from Sorghum bicolor.

We assessed the effects of chloroform extract of fermented Viola mandshurica (CEFV) on melanogenesis B16 melanoma cells. CEFV treatment significantly decreased melanin content and tyrosinase activity in dose-dependent manners. To elucidate the mechanism of the inhibitory effects of CEFV on melanogenesis, we performed RT-PCR and Western blotting for melanogenesis-related genes such as tyrosinase, tyrosinase-related protein-1 (TRP-1), TRP-2, and microphthalmia-associated transcription factor (MITF). CEFV strongly inhibited mRNA as well as the protein expression of tyrosinase and MITF, but had no significant effect on TRP-1 or TRP-2 expressions. It markedly decreased the phosphorylation of cAMP responsive element binding protein (CREB), and induced the duration of extracellular signal-regulated kinase (ERK) activation, leading to reduction of MITF expression and subsequently that of tyrosinase. Therefore, we suggest that CEFV induces downregulation of melanogenesis through decreased CREB phosphorylation and ERK activation.

Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.

The butanol-soluble fraction of the dried root of Angelica gigas exhibited significant protection against chloroquine-sensitive strains of Plasmodium falciparum using the parasite lactate dehydrogenase assay method. Using antiplasmodial activity-guided fractionation, five coumarins, marmesinin (1), nodakenin (2), skimmin (3), apiosylskimmin (4), and magnolioside (5), were isolated and evaluated for in vitro antiplasmodial activity, as well as for their cytotoxic potential on SK-OV-3 cancer cell lines. Compounds 1 and 5 showed notable growth inhibitory activity against chloroquine-sensitive strains of P. falciparum with IC(50) values of 5.3 and 8.2 μM. The compounds showed no significant cytotoxicity (IC(50) > 100 μM) toward the SK-OV-3 cancer cell line. This is the first report on the antiplasmodial activity of these coumarin derivatives from the dried root of A. gigas.

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 μM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.

National Academy of Agricultural Science, RDA, Suwon 441-707, Korea (Received on August 10, 2010; Accepted on October 26, 2010)Suppression subtractive hybridization was used toisolate wound-induced genes from soybean. One of thewound-induced genes, gmwi143 designated as GmCCR,showed high homology with genes encoding cinnamoyl-CoA reductase (CCR; EC 1.2.1.44). Deduced aminoacid sequences encoded by GmCCR showed the highestidentity (77%) with those of Acacia CCR. There are 2CCR genes highly homologous to GmCCR in soybeangenome based on Phytozome DB analysis. RNA expre-ssion of GmCCR was specifically induced by local andsystemic wounding, drought, high salinity or by ultra-violet stress. Our study suggests that GmCCR may beinvolved in resistance mechanism during abiotic stressesin plants. Keywords :cinnamoyl-coA reductase, lignin, soybean, wound-ing, UVLignin, a major component of secondary cell walls, isdeposited mainly in the vascular tissues during plantdevelopment and environmental signals (Boerjan et al.,2003). Lignins contribute to major functions in plants, suchas stem rigidity, water transport in xylem and are alsoinvolved in defense reactions against wounding, predatorsor pathogens (Vance et al., 1980). It has been reported thatlignin biosynthesis is regulated by developmental signal inthe vascular tissues and by defense responses in plant(Lauvergeat et al., 2001).The biosynthesis of lignins begins with the commonphenylpropanoid pathway, starting from phenylalanine andleading to cinnamoyl CoAs which are the common pre-cursors of a wide range of phenolic compounds. These CoAesters subsequently are changed to monolignols via tworeductive steps catalyzed by cinnamoyl CoA:NADP oxido-reductase (CCR, EC 1.2.1.44) and cinnamyl alcohol de-hydrogenase (CAD, EC 1.1.1.195). Lignins result from theoxidative polymerization of monolignols. Several genes encoding CCR have been shown to betranscriptionally induced by developing xylem tissues or bybiotic stress (Kim et al., 2000; Lacombe et al., 1997;Lauvergeat et al., 2001). Lacombe et al. (1997) showed thatEucalyptus CCR transcript was expressed in lignified organssuch as leaves, stems and roots. By in situ hybridizationtechnique, EuCCR was strongly expressed in the differ-entiating xylem zone but was not present in the secondaryphloem fibers or in the young periderm (Lacombe et al.,1997). Rice OsCCR1 RNA expression was induced insuspension cell cultures treated with sphingolipid elicitorpurified from rice blast Magnaporthe grisea (Kawasaki etal., 2006). Previously, soybean chip analysis showed thatRNA expression of CCR invovled in phenylpropanoidpathway were induced in the syncytia laser microdissectedsoybean roots infected with soybean cyst nematode (Klinket al., 2009).There are 11 putative CCR homologues sharing homo-logy identities raging from 82.8 to 32.8% in Arabidopsisgenome (Costa et al., 2003). Arabidopsis AtCCR1 transcriptwas expressed in the stem and floral tissues (Lauvergeat etal., 2001) and the irregular xylem4 mutant (irx4) defectivein the AtCCR1 gene showed severely reduced lignin con-tents (50%) compared to the wild type (Jones et al., 2001).Lauvergeat et al. (2001) suggested that AtCCR2 responsiveto pathogen infection of Xanthomonas campestris pv.campestris may play a role in the formation of phenoliccompounds associated with HR in Arabidopsis (Lauvergeatet al., 2001). In the present study, we have isolated the full-lengthcDNA of GmCCR encoding CCR-like protein regulated byabiotic stresses. We propose that the GmCCR is involved indefense responses during environmental stresses in plants.

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 μmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 μM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 μM.

Aims: The aim of this study was to isolate and characterize new bacteriophages that infect a wide range of plant pathogenic Ralstonia solanacearum strains. Methods and Results: Fifteen bacteriophages were isolated from pepper, tomato and tobacco plant rhizospheres infected with R. solanacearum. A host specificity analysis of the isolated phages using nine strains of R. solanacearum indicated great phage diversity in a single soil. Two phages, PE226 and TM227, showed clear plaques on all nine bacterial hosts tested and were virtually identical in morphology and genome. PE226, an Inovirus, is a long, flexible, filamentous phage carrying a circular (+) sense single-strand DNA genome of 5475 nucleotides. DNA sequences of PE226 exhibited nine open reading frames (ORF) that were not highly similar to those of other phages infecting R. solanacearum. The genome organization of PE226 was partially similar to that of p12J of Ralstonia pickettii. One ORF of PE226 showed identity to the zot gene encoding zonula occludens toxin of Vibrio cholera. Orf7 of PE226 was also present in the genome of R. solanacearum strain SL341. However, SL341, a highly virulent strain in tomato, was still sensitive to phage PE226. Conclusions: A new, flexible, filamentous phage PE226 infected wide range of R. solanacearum strains and carried unique circular single-strand DNA genome with an ORF encoding Zot-like protein. Significance and Impact of the Study: PE226 may be a new type of temperate phage, based on its lytic nature on a wide range of hosts and the presence of a zot homologue in a host bacterial genome.

Plant secondary metabolites have always been a focus of study due to their important roles in human medicine and nutrition. We transferred the isoflavone synthase (IFS) gene into soybean [Glycine max (L.) Merr.] using the Agrobacterium-mediated transformation method in an attempt to produce transformed soybean plants which produced increased levels of the secondary metabolite, isoflavone. Although the trial to produce transgenic plant failed due to unestablished hygromycin selection, transformed callus cell lines were obtained. The induction rate and degree of callus were similar among the three cultivars tested, but light illumination positively influenced the frequency of callus formation, resulting in a callus induction rate of 74% for Kwangan, 67% for Sojin, and 73% for Duyou. Following seven to eight subcultures on selection media, the isoflavone content of the transformed callus lines were analyzed by high-performance liquid chromatography. The total amount of isoflavone in the transformed callus cell lines was three- to sixfold higher than that in control callus or seeds. Given the many positive effects of isoflavone on human health, it may be possible to adapt our transformed callus lines for industrialization through an alternative cell culture system to produce high concentrations of isoflavones.

Lesion mimic mutant (LMM) plants display spontaneous necrotic lesions on their leaves without any pathogenic infection. Specific rice LMMs designated as spotted leaf (spl) including spl1, spl3, spl4, spl5 and spl6 are genetically known as lesion resembling disease (lrd) mutant. The inheritance patterns in the and progenies of these mutants are controlled by recessive genetic factors. Lesion development in the rice LMMs were controlled by both development stages and environmental factors. The rice LMMs exhibited higher numbers of spots under temperature than those under . Contents of chlorophyll were drastically reduced at 60 days old LMM leaves when the spot formation was severe. The levels of endogenous hydrogen peroxide were highest at 45 days old mutants but reduced at 60 days old. Transcription levels of stress related genes including thioredoxin peroxidase and protein disulfide isomerase were reduced in spotted leaves than those of non spotted leaves. It could be suggested that scavenging system against reactive oxygen species induced by either stresses or innate metabolisms may not work properly in the rice LMMs. As these rice LMMs autonomously expressed clear lesions of lrd phenotype without pathogen infection, it could be useful to understand stresses responses in plants.

Our previous study has demonstrated that the methanol extract of Hyul-Tong-Ryung (HM) specifically suppresses the phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) production through the inhibition of MMP-9 mRNA expression in MCF-7 human breast carcinoma cells. However, the molecular mechanisms involved in transcriptional suppression of MMP-9 by HM in PMA-induced MCF-7 cells are not known. In this study, we aimed to elucidate the molecular mechanisms involved in the inhibition of MMP-9 expression by HM in PMA-induced MCF-7 cells. The results of promoter assay and EMSA showed that HM specifically inhibits MMP-9 gene expression by blocking PMA-stimulated activation of activator protein-1 (AP-1). In addition, PMA-stimulated phosphorylation of extracellular signal regulated kinase 1/2 (ERK 1/2) was suppressed by HM treatment, whereas the phosphorylation of either c-Jun N-terminal kinase (JNK) or p38 mitogen-activated protein kinase (MAPK) was not affected. HM could inhibit the PMA-induced MMP-9 expression through suppression of the transcriptional activity of MMP-9 gene in MCF-7 cells. These results indicate that HM inhibits PMA-induced MMP-9 expression by blocking the activation of activator protein-1 (AP-1) via extracellular signal regulated kinase 1/2 (ERK 1/2) signaling pathway.

The present study examined the effect of the king oyster mushroom (Pleurotus eryngii) on insulin resistance and dyslipidemia in db/db mice. Four-week-old db/db mice were fed an AIN-93G diet or a diet containing 5% king oyster mushroom for 7 weeks. The blood glycated hemoglobin and serum glucose levels in the mushroom group were significantly lower than the control group (p

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Hydrolytic conditions of rice hulls by acid and alkaline treatments before enzymatic saccharification were optimized in this study. Based on the results of single-factor experiments and an orthogonal array experiment, reaction time was found to be the most important factor for the acidic hydrolysis of rice hulls. Maximum yield of sugars from 1 g of rice hulls by acidic treatment under optimized conditions was 213.6 mg. The yield of lignin removal from acidic pretreated rice hulls by alkaline treatment increased with increase in reaction temperature and time. The amount of sugars obtained from 1 g of pretreated rice hulls by enzymatic saccharification was 307.7 mg, and the conversion rate of sugars from crude fibers in pretreated rice hulls was about 72%. Instrumental analyses with FTIR and SEM indicated that lignin in rice hulls was partially removed by alkaline treatment, and the structure of rice hulls became deformed and more fibers were exposed to cellulases after acidic treatment.

A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60–63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P i ). The induced expression of OsPAP2 is a specific response to P i starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P i -supply, and transcripts of OsPAP2 were abundant in P i -deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P i -deficient conditions.

The translation elongation factor 1A, eEF1A, catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome by a GTP-dependent mechanism. By subtractive suppression hybridization technique, we have isolated a soybean low-temperature inducible gene, SLTI100 encoding translation elongation factor 1A. Multiple sequence align-ments and phylogenic analysis showed that SLTI100 and other eEF1As originated from diverse organisms are highly conserved. RNA expression of SLTI100 was specifically induced by low temperature, high salt, ABA, or drought stress. Based on the subcellular localization of the corre-sponding gene product fused to GFP, we were able to con-firm that SLTI100-GFP was restricted to the nucleus and cytoplasm. We propose that soybean eEF1A may play an important role in translational regulation during abiotic stress responses in plants. Introduction In the translational elongation process, eukaryote transla-tional elongation factor 1 (eEF1) is a protein synthesis factor composed of four subunits: A, B α, Bβ and Bγ (Browing et al. 1990). Subunit eEF1A, also known as EF1α, binds ami-noacyl-tRNAs to the acceptor (A) site of the ribosome during the peptide chain elongation phase of protein syn-thesis. eEF1A (49,174 kDa) is an abundant protein repre-senting upto 5% of the soluble protein in wheat germ extracts (Browing et al. 1990) and is highly conserved in evolution (77% conservation of wheat EF1A with human EF1A) (Metz et al. 1992). In higher eukaryotes, eEF1A is typically encoded by a multigene family: in man there are 18 genes (Lund et al. 1996) while in

We isolated and characterized a stress-inducible gene, designated as Slti114, encoding matrix metalloproteinase (MMP) in soybean. The derived amino acid sequences of Slti114 show the 69 % homology with MMP2 from Glycine max (AAL27029). The size of the full-length cDNA of Slti114 is 1194 bp with open reading frame comprised of 394 amino acids. RNA expression of Slti114 was induced by low temperature or wounding. During early stage, Slti114 RNA level was extremely high, but Slti114 RNA was not detectable just after cotyledons became yellowish. Green fluorescent protein fusion expression system confirmed that Slti114-smGFP and H+-ATPase-RFP were co-localized to the plasma membrane. Purified glutathione-S-transferase (GST)-Slti114 protein was shown to digest myelin basic protein (MBP) in vitro, but not gelatin. This report provides strong evidence that plasma membrane MMP, Slti114 protein may play a critical role during abiotic stress and senescence in plant.

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH((1-355))-GFP is localized in the nucleus, whereas GmRH((130-355))-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.

We isolated the full-length genomic clone of STLI25 encoding RPS13 (ribosomal protein S13) in soybean. Genomic DNA structure of SLTI25 is similar to that of Arabidopsis ribosomal protein S13. RNA expression of SLTI25 was induced by salt, ABA, or wounding stress, but reduced by dehydration stress. To determine the subcellular localization of the gene product fused to GFP, we were able to confirm that SLTI25-GFP was restricted to the nucleus. By domain swapping analysis, it was shown that C-terminal region of SLTI25 with a putative nucleus localization signal was necessary and sufficient for nucleus targeting of the fusion protein in plants. This new findings provide an evidence that SLTI25 is targeted to the nucleus for the ribosome subunit assembly in plants.

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants.

We identified the promoter region of the human GD3 synthase (hST8Sia I) gene to elucidate the mechanism underlying the regulation of hST8Sia I expression in human glioblastoma cells. The 5'-rapid amplification of cDNA end using mRNA prepared from U-87MG cells revealed the presence of transcription start site of hST8Sia I gene, and the 5'-terminal analysis of its product showed that transcription started from 648 nucleotides upstream of the translational initiation site. Functional analysis of the 5'-flanking region of the hST8Sia I gene by transient expression method revealed that the region from -638 to -498 is important for transcriptional activity of the hST8Sia I gene in U-87MG and T98G cells. This region lacks apparent TATA and CAAT boxes, but contains putative binding sites for transcription factors AREB6 and Elk-1. Site-directed mutagenesis and transient transfection assays demonstrated that both AREB6 and Elk-1 elements in this region were required for the promoter activity in U-87MG and T98G cells. These results indicated that both AREB6 and Elk-1 might play an essential role in the transcriptional activity of hST8Sia I gene essential for GD3 synthesis in human glioblastoma cells.

Photorespiration reduces carbon fixation rate, but it is an essential process in plant. Photorespiration involves reactions in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes the conversion of alanine and glyoxylate into glycine and pyruvate, respectively. We isolated a low temperature-inducible cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated asMLT92, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed that the mungbean genome has one copy ofMLT92. MLT92 mRNA was induced by low temperature and its RNA expression was not much increased by drought stress. ABA and NaCl did not induce RNA expression ofMLT92. Based on GFP/RFP targeting experiment, GFP-MLT92 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome in tobacco protoplasts. This suggests that peroxisomalMLT92 is involved in molecular response to low temperature stress.

secreted from Bacillus sp. J105 strain was purified to a single band on SDS-PAGE by ammonium sulfate precipitation, ion exchange column chromatography and gel-filtration. The molecular weight of the purified enzyme was 31 kDa on SDS-PAGE and its isoelectric point was 7.35. Optimal pH and temperature for enzymatic reaction were 5 and , respectively. As a result of total amino acid composition analysis of the purified enzyme, Gly and Ala were occupied 14.1 and 13.3 mole %, respectively. Km and Vmax value of purified enzyme were 1.33 mM and 0.36 mM/ml using ampicillin as a substrate, respectively.

Cotyledons of perilla6 were cultured on MS medium containing 0.5 mg/l NAA and 0.5 mg/l BA for 7 weeks. The activity of perilla peroxidase was observed to increase following culture stages as assessed by peroxidase assay. A peroxidase (POD) was purified from perilla tissue cultured on MS medium for 7 weeks. The peroxidase was purified using ion exchange and gel nitration chromatography. The perilla peroxidase had a molecular mass of 30 kDa by SDS-PAGE. We showed that the N-terminal amino acid sequence of this protein shared 67% identity with the tea peroxidase. As indicated by SDS-PAGE, the banding pattern of the 30 kDa polypeptide present in total soluble protein from perilla tissue was increased following culture stages. Immunoblot analysis indicated that perilla peroxidase protein appeared after 3 weeks of perilla tissue culture, and continued to increase with extended duration of tissue culture for at least 7 weeks.