Youn-Hyung Lee

A comprehensive expression analysis of the WRKY gene superfamily in rice plants during defense response

To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens.

Role of the Rice Hexokinases OsHXK5 and OsHXK6 as Glucose Sensors

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Molecular cloning and expression analysis of the cell-wall invertase gene family in rice ( Oryza sativa L.)

Cell-wall invertase (CIN) catalyzes the hydrolysis of sucrose into glucose and fructose for the supply of carbohydrates to sink organs via an apoplastic pathway. To study the CIN genes in rice (Oryza sativa L.), we isolated cDNA clones showing amino acid similarity to the plant cell wall invertase proteins from a search of rice sequence databases. Profile analyses revealed that the cloned genes are expressed in unique patterns in various organs. For example, transcripts of OsCIN1, OsCIN2, OsCIN4, and OsCIN7 were detected in immature seeds whereas OsCIN3 gene expression was flower-specific. Further transcript analysis of these genes expressed in developing seeds indicated that OsCIN1, OsCIN2, and OsCIN7 might play an important role involving sucrose partitioning to the embryo and endosperm. Sucrose, a substrate of CINs, induced the accumulation of OsCIN1 transcripts in excised leaves and OsCIN2 in immature seeds, while the level of OsCIN5 was significantly down-regulated in excised leaves treated with sucrose. Infecting the tissues with rice blast (Magnaporthe grisea) as a biotic stressor increased the expression of OsCIN1, OsCIN4, and OsCIN5, suggesting that these genes may participate in a switch in metabolism to resist pathogen invasion. These results demonstrate that OsCIN genes play diverse roles involving the regulation of metabolism, growth, development, and stress responses.

Structure, expression, and functional analysis of the hexokinase gene family in rice (Oryza sativa L.)

Hexokinase (HXK) is a dual-function enzyme that both phosphorylates hexose to form hexose 6−phosphate and plays an important role in sugar sensing and signaling. To investigate the roles of hexokinases in rice growth and development, we analyzed rice sequence databases and isolated ten rice hexokinase cDNAs, OsHXK1 (Oryza sativa Hexokinase 1) through OsHXK10. With the exception of the single-exon gene OsHXK1, the OsHXKs all have a highly conserved genomic structure consisting of nine exons and eight introns. Gene expression profiling revealed that OsHXK2 through OsHXK9 are expressed ubiquitously in various organs, whereas OsHXK10 expression is pollen-specific. Sugars induced the expression of three OsHXKs, OsHXK2, OsHXK5, and OsHXK6, in excised leaves, while suppressing OsHXK7 expression in excised leaves and immature seeds. The hexokinase activity of the OsHXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). OsHXK4 was able to complement this mutant only after the chloroplast-transit peptide was removed. The subcellular localization of OsHXK4 and OsHXK7, observed with green fluorescent protein (GFP) fusion constructs, indicated that OsHXK4 is a plastid-stroma-targeted hexokinase while OsHXK7 localizes to the cytosol.

Dynamic sensing using intelligent composite: an investigation to development of new pH sensors and electrochromic devices

Abstract A composite of two conducting polymers, polyaniline and polypyrrole, could result in much improved physical properties with respect to their applications in electrochromic devices. However, the composite has time dependant properties that made the use of dynamic computational algorithms necessary to control its behaviour. We used a platinum modified electrode as an electrochromic display device, which was integrated with a pre-trained computer for data processing and knowledge discovery. Since there is a direct relationship between the colour of the composite and its conductivity, detection of conductivity/colour has been used for prediction of pH, we have found that different parameters within the algorithms can affect the performance of our modelling resulting in a more reliable device.

Droplet merging in a straight microchannel using droplet size or viscosity difference

In the present study, a novel droplet-merging technique is proposed, which enables two nanoliter or picoliter droplets to merge in a straight microchannel with high precision. Two dispersed phase fluids are supplied respectively from two side channels in a certain range of flow rates to generate droplets of different sizes or different viscosities in a regularly alternating mode at a cross-channel intersection prior to the straight microchannel, in such a manner that the droplets are spontaneously synchronized by themselves due to the competition of the two interfaces formed at the cross-channel intersection. Then they are autogeneously merged in the downstream straight microchannel with or without a sudden expansion of the cross section, due to their velocity difference which is induced as they are transported aboard the continuous phase fluid. This droplet-merging method has no desynchronization or secondary-merging problems. Thus, it can be applied efficiently to mixing or encapsulating one target sample with another material for the purposes of nanoparticle synthesis, hydrogel-bead production, cell transplantation and so forth.

Ergosterol Peroxide from Flowers of Erigeron annuus L. as an Anti-Atherosclerosis Agent

Flowers of Erigeron annuus L. were extracted with 80% aqueous MeOH, and the concentrated extract was partitioned with EtOAc, n-BuOH, and H2O. Repeated silica gel and ODS column chromatography of the EtOAc fraction led to the isolation of a sterol, through activity-guided fractionation, using ACAT inhibitory activity measurements. From the physico-chemical data, including NMR, MS, and IR, the chemical structure of the compound was determined to be an ergosterol peroxide (1), which has been isolated for the first time from this plant. This compound exhibited hACAT-1 and Lp-PLA2 inhibitory effects, with inhibitory values of 51.6 ± 0.9 and 51.7 ± 1.2%, at a treatment concentration of 0.23 mM.

Synthesis and characterization of the first adamantane-based poly(p-phenylenevinylene) derivative: an intelligent plastic for smart electronic displays

Abstract A novel poly( p -phenylenevinylene) (PPV) derivative containing adamantane substituent—poly(2-methoxy-5-adamantaneethyloxy- p -phenylenevinylene) (MAE-PPV) is synthesized through the Gilch route. Adamantane was incorporated into this polymer because of its rigid and spherical structure, which results in reduction of interchain interaction. MAE-PPV is solution processable and shows high photoluminescence intensity. This polymer also demonstrates good electroluminescene properties.

Purification and characterization of a recombinant pea cytoplasmic fructose-1,6-bisphosphatase.

Full-length cDNA encoding pea cytoplasmic fructose-1,6-bisphosphatase (cyFBPase) was cloned from a pea cDNA library. The cloned cDNA was introduced into the Escherichia coli expression vector pET-15b. The recombinant cyFBPase was expressed in E. coli BL21 (DE3) cells in a soluble form and purified to homogeneity by Ni(+)-NTA affinity chromatography. The identity of the recombinant cyFBPase was confirmed by SDS-PAGE and immunoblot analysis using a polyclonal anti-His tag antibody. The recombinant cyFBPase was active at neutral pH ranges (6.6-9.0) and thermostable as other cyFBPases. The activation energy (E(a)) and Arrhenius frequency factor were 17.4 kcal/mol and 2.6 x 10(12)/s, respectively. The K(M) and V(max) values of the recombinant enzyme were calculated as 10.47 microM and 109 micromol/min, respectively. In case of removal of histidine tag, the K(M) value was calculated as 5.03 microM. The recombinant enzyme was non-competitively and competitively inhibited by AMP and fructose-2,6-bisphosphate, respectively.

Examination of EL and PL properties of MCHM-PPV and MEH-PPV: a study towards introduction of a new series of thin film EL devices

Abstract The effect of the side group, employed during synthesis, on the PL (photoluminescence), EL (electroluminescence) profiles and the light intensity of PPV derivatives has been investigated using MEH (methoxy-ethyl-hexyloxy) and MCHM (methoxy-cyclo-hexyl-methyloxy) as side groups. The measurements show that even the most subtle change in molecular structure of PPV has a marked improvement on the EL, PL profiles and the light emission intensity. It has been found that the PL and EL intensities of the MCHM-PPV are higher than those of MEH-PPV. MCHM-PPV also demonstrated higher light emission at lower potentials and currents.