Jongbum Park

ATHB12, an ABA-Inducible Homeodomain-Leucine Zipper (HD-Zip) Protein of Arabidopsis, Negatively Regulates the Growth of the Inflorescence Stem by Decreasing the Expression of a Gibberellin 20-Oxidase Gene

Arabidopsis thaliana homeobox 12 (ATHB12) is rapidly induced by ABA and water stress. A T-DNA insertion mutant of ATHB12 with a reduced level of ATHB12 expression in stems had longer inflorescence stems and reduced sensitivity to ABA during germination. A high level of transcripts of gibberellin 20-oxidase 1 (GA20ox1), a key enzyme in the synthesis of gibberellins, was detected in athb12 stems, while transgenic lines overexpressing ATHB12 (A12OX) had a reduced level of GA20ox1 in stems. Consistent with these data, ABA treatment of wild-type plants resulted in decreased GA20ox1 expression whereas ABA treatment of the athb12 mutant gave rise to slightly decreased GA20ox1 expression. Retarded stem growth in 3-week-old A12OX plants was rescued by exogenous GA(9), but not by GA(12), and less GA(9) was detected in A12OX stems than in wild-type stems. These data imply that ATHB12 decreases GA20ox1 expression in stems. On the other hand, the stems of A12OX plants grew rapidly after the first 3 weeks, so that they were almost as high as wild-type plants at about 5 weeks after germination. We also found changes in the stems of transgenic plants overexpressing ATHB12, such as alterations of expression GA20ox and GA3ox genes, and of GA(4) levels, which appear to result from feedback regulation. Repression of GA20ox1 by ATHB12 was confirmed by transfection of leaf protoplasts. ABA-treated protoplasts also showed increased ATHB12 expression and reduced GA20ox1 expression. These findings all suggest that ATHB12 negatively regulates the expression of a GA 20-oxidase gene in inflorescence stems.

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain‐leucine zipper protein, regulates leaf growth by promoting cell expansion and endoreduplication

Arabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HD-Zip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure. To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) - P35 S ::ATHB12SRDX (A12SRDX) and PATHB 12 ::ATHB12SRDX - slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated. Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4). Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis.

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.

Purification and Biochemical Characterization of Insoluble Acid Invertase (INAC-INV) from Pea Seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.

Biochemical Characterization of Soluble Acid and Alkaline Invertases from Shoots of Etiolated Pea Seedlings

Soluble invertase was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, DEAE-Sepharose column, Con-A- and Green 19-Sepharose affinity columns, hydroxyapatite column, ultra-filtration, and Sephacryl 300 gel filtration. The purified soluble acid (SAC) and alkaline (SALK) invertases had a pH optimum of 5.3 and 7.3, respectively. The temperature optimum of two invertases was 37 degrees C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the two purified enzymes were examined. Tris-HCl and HgCl(2) did not affect SAC activity, whereas 10 mM Tris-HCl and 0.05 mM HgCl(2) inhibited SALK activity by about 50%. SAC and SALK were inhibited by 4.8 mM and 0.6 mM CuSO(4) by 50%, respectively. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The Kms of SAC and SALK were determined to be 1.8 and 38.6 mM, respectively. The molecular masses of SAC shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were 22 kDa and 45 kDa. The molecular mass of SALK was 30 kDa. Iso-electric points of the SAC and SALK were estimated to be about pH 7.0 and pH 5.7, respectively.

Analysis of Biochemical Compositions and Nutritive Values of Six Species of Seaweeds

The biochemical compositions and nutritive values of six species of seaweeds were analyzed to determine their applicability in functional foods or ingredients. The biochemical compositions (moisture, ash, protein, lipid, and dietary fiber) and fatty acid contents were determined for the following seaweed extracts: Phaeophyceae (Laminaria japonica, Hizikia fusiformis, and Undaria pinnatifida), Rhodophyceae (Porphyra tenera and Gracilaria verrucosa), and Chlorophyceae (Ulva lactuca). The moisture content (% dry weight) ranged from 11.47% to 13.94%, ash from 19.15% to 26.50%, protein from 5.08% to 15.44%, lipid from 2.75% to 4.43%, and dietary fiber from 36.84% to 52.98%. C14:0, C16:0, C18:0, C16:1, C18: 1n-3, C18:2n-6, C18:3n-6, C20:4n-6, and C20:5n-3 represented the predominant proportions of fatty acids. Interestingly, docosahexaenoic acid (C22:6n-3, DHA) was either not found or only detected in trace amounts in the analyzed seaweeds. The levels of n-3 fatty acid were higher than other polyunsaturated fatty acids, and the n-6/n-3 ratio was very low. These results indicate that seaweed inhabiting Korean coastal areas will be beneficial to human health.

Phenolic Contents and Antioxida nt Activities of Six Edible Seaweeds

Abstract Phenolic contents and antioxidant activities were determined in the water and ethanol extracts from six species ofedible seaweeds, Phaeophyceae ( Laminaria japonica, Hizikia fusiformis and Undaria pinnatifida ), Rhodophyceae ( Porphyra tenera and Gracilaria verrucosa ) and Chlorophyceae ( Ulva lactuca ). The highest extraction yield was observed in water extract (pH 8.0) of G. verrucosa (44.23 %) and phenolic content was the highest in ethanolic (75%) extract of H. fusiformis (52.82 μg/mg). 25 % ethanolic extract from U. lactuca was found to have the highest DPPH radicals scavenging activity(19.29 %) and superoxide anion scavenging activitiy was higher in water extract (pH 8.0) from H. fusiformis (81.20 %) than that other seaweeds. Phenolic contents were strongly correlated with antioxidant activity in the six edible seaweeds extracts (R 2 =0.852). Taken together, these results indicate that H fusiformis may be a excellentsource for development of natural antioxidants. Key Words :

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

Phenolic Content, DPPH Radical Scavenging, and Tyrosinase Inhibitory Activities of Ecklonia cava Extracted with the Ultrasonic Wave Method

The applicability of the ultrasonic wave method to the extraction of useful components from seaweeds was investigated. Extracts from freeze-dried Ecklonia cava powder were prepared with hot water (65°C), water (24°C), 50% ethanol, and 100% ethanol, and ultrasonic extraction was also performed. The content of phenolic compounds and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and tyrosinase inhibitory activity of the extracts were analyzed, and differences in the data obtained by the ultrasonic extraction and the traditional extraction methods were compared. The phenolic content in the E. cava extract by ultrasonic extraction (142.80 mg/g) was approximately 14 times higher than the phenolic content in the hot water extract (10.03 mg/g). The DPPH radical scavenging and the tyrosinase inhibitory activities of the ultrasonic extract were approximately 4 times and 14 times higher than the hot water extracts, respectively. The correlation between the phenolic content and the DPPH radical scavenging activity (R2=99.47) and between the phenolic content and the tyrosinase inhibitory activity (R2=99.99) was very high. These results indicate that ultrasonic extraction is more suitable than traditional extraction for the extraction of useful components from E. cava.

Production of Red-spotted Grouper Nervous Necrosis Virus (RGNNV) Capsid Protein Using Saccharomyces cerevisiae Surface Display

Protein Using Saccharomyces cerevisiae Surface Display Mirye Park, Sung-Suk Suh, Jinik Hwang, Donggiun Kim, Jongbum Park, Young-Jae Chung and Taek-Kyun Lee 1 * South Sea Environment Research Department, Korea Institute of Ocean Science and Technology, Geoje 656-830, Korea Department of Biological Science, Silla University, Busan 617-736, Korea Department of Life Science and Biotechnology, Shin Gyeong University, Hwaseong 445-741, KoreaThe studies of marine viruses in terms of viral isolation and detection have been limited due to the high mutation rate and genetic diversity of marine viruses. Of the modern methods currently used to detect marine viruses, serological methods based on enzyme-linked immunosorbent assay (ELISA) are the most common. They depend largely on the quality of the antibodies and on highly purified suitable antigens. Recently, a new experimental system for using viral capsid protein as an antigen has been developed using the yeast surface display (YSD) technique. In the present study, the capsid protein gene of the red-spotted grouper nervous necrosis virus (RGNNV) was expressed and purified via YSD and HA-tagging systems, respectively. Two regions of the RGNNV capsid protein gene, RGNNV1 and RGNNV2, were individually synthesized and subcloned into a yeast expression vector, pCTCON. The expressions of each RGNNV capsid protein in the Saccharomyces cerevisiae strain EBY100 were indirectly detected by flow cytometry with fluorescently labeled antibodies, while recognizing the C-terminal c-myc tags encoded by the display vector. The expressed RGNNV capsid proteins were isolated from the yeast surface through the cleavage of the disulfide bond between the Aga1 and Aga2 proteins after β-mercaptoethanol treatment, and they were directly detected by Western blot using anti-HA antibody. These results indicated that YSD and HA-tagging systems could be applicable to the expressions and purification of recombinant RGNNV capsid proteins.