Hyeonsook Cheong

Secretome Analysis Reveals an Arabidopsis Lipase Involved in Defense against Alternaria brassicicola

The Arabidopsis thaliana secretome was analyzed by the proteomic approach, which led to the identification of secreted proteins implicated in many aspects of cell biology. We then investigated the change in the Arabidopsis secretome in response to salicylic acid and identified several proteins involved in pathogen response. One of these, a secreted lipase with a GDSL-like motif designated GDSL LIPASE1 (GLIP1), was further characterized for its function in disease resistance. glip1 plants were markedly more susceptible to infection by the necrotrophic fungus Alternaria brassicicola compared with the parental wild-type plants. The recombinant GLIP1 protein possessed lipase and antimicrobial activities that directly disrupt fungal spore integrity. Furthermore, GLIP1 appeared to trigger systemic resistance signaling in plants when challenged with A. brassicicola, because pretreatment of the glip1 mutant with recombinant GLIP1 protein inhibited A. brassicicola–induced cell death in both peripheral and distal leaves. Moreover, glip1 showed altered expression of defense- and ethylene-related genes. GLIP1 transcription was increased by ethephon, the ethylene releaser, but not by salicylic acid or jasmonic acid. These results suggest that GLIP1, in association with ethylene signaling, may be a critical component in plant resistance to A. brassicicola.

A putative novel transcription factor, AtSKIP, is involved in abscisic acid signalling and confers salt and osmotic tolerance in Arabidopsis

We identified and functionally characterized the AtSKIP gene (At1g77180), an Arabidopsis homologue of SNW/SKIP, under abiotic stresses. Although the SNW/SKIP protein has been implicated as a critical transcription cofactor, its biological functions have yet to be reported in any plant. Recently, we have isolated Salt-tolerance genes (SATs) via the overexpression screening of yeast with a maize cDNA library. One of the selected genes (SAT2) appeared to confer elevated tolerance to salt. Maize SAT2 cDNA encodes a homologue of the human SNW/SKIP transcriptional coregulator. Treatment with salt, mannitol and abscisic acid induced AtSKIP expression. Ectopic expression of the AtSKIP gene modulated the induction of salt tolerance, dehydration resistance and insensitivity towards abscisic acid under stress conditions. By contrast, atskip antisense lines displayed reduced tolerance to abiotic stresses during germination. Moreover, a decrease in AtSKIP expression resulted in an abnormal phenotype. We further determined that the AtSKIP protein activated the transcription of a reporter gene in yeast. Green fluorescent protein-tagged AtSKIP was localized in the nuclei of both onion cells and transgenic Arabidopsis cells. Taken together, these results suggest that AtSKIP functions as both a positive regulator and putative potential transcription factor in the abiotic stress signalling pathway.

Molecular and Physiological Characterization of the Arabidopsis thaliana Oxidation-Related Zinc Finger 2, a Plasma Membrane Protein Involved in ABA and Salt Stress Response Through the ABI2-Mediated Signaling Pathway

CCCH-type zinc finger proteins are important for developmental and environmental responses. However, the precise roles of these proteins in plant stress tolerance are poorly understood. Arabidopsis thaliana Oxidation-related Zinc Finger 2 (AtOZF2) (At4g29190) is an AtOZF1 homolog previously isolated from Arabidopsis, which confers oxidative stress tolerance on plants. The AtOZF2 protein is localized in the plasma membrane, as is AtOZF1. Disruption expression of AtOZF2 led to reduced root length and leaf size. AtOZF2 was implicated to be involved in the ABA and salinity responses. atozf2 antisense lines were more sensitive to ABA and salt stress during the seed germination and cotyledon greening processes. In contrast, AtOZF2-overexpressing plants were more insensitive to ABA and salt stress than the wild type. Interestingly, in the presence of ABA and salt stress, the transcript level of ABA insensitive 2 (ABI2), but not that of ABI1, in AtOZF2-overexpressing plants was lower than that in the wild type, whereas the expression of ABI2 in atozf2 was significantly enhanced. Thus, AtOZF2 is involved in the ABA and salt stress response through the ABI2-mediated signaling pathway. Taken together, these findings provide compelling evidence that AtOZF2 is an important regulator for plant tolerance to abiotic stress.

Physiological characterization of the Arabidopsis thaliana Oxidation-related Zinc Finger 1, a plasma membrane protein involved in oxidative stress

The CCCH-type zinc finger proteins are a superfamily containing tandem zinc-binding motifs involved in many aspects of plant growth and development. However, the precise role of these proteins involved in plant stress tolerance is poorly understood. This study was to examine the regulatory and functional role of the CCCH-type zinc finger protein, AtOZF1 (At2g19810), under oxidative stress. Interestingly, the AtOZF1 protein was localized in the plasma membrane. The AtOZF1 transcripts were highly induced by treatment with hydrogen peroxide, abscisic acid and salinity. The AtOZF1-overexpressing plants were relatively resistant to oxidative stress than wild-type and T-DNA insertion mutant atozf1. Malondialdehyde, a decomposition product of lipid peroxidation, accumulated in atozf1 mutants more than in wild-type and AtOZF1-overexpressing plants. Furthermore, atozf1 mutants displayed lower activities of catalase and guaiacol peroxidase, higher chlorosis, and down-regulated expression of antioxidant genes under oxidative stress. Taken together, these observations demonstrate that AtOZF1 is required for the tolerance of Arabidopsis to oxidative stress.

Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens

A novel antifungal protein (Pr-2) was identified from pumpkin rinds using water-soluble extraction, ultrafiltration, cation exchange chromatography, and reverse-phase high-performance liquid chromatography. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicated that the protein had a molecular mass of 14865.57 Da. Automated Edman degradation showed that the N-terminal sequence of Pr-2 was QGIGVGDNDGKRGKR-. The Pr-2 protein strongly inhibited in vitro growth of Botrytis cinerea, Colletotrichum coccodes, Fusarium solani, Fusarium oxysporum, and Trichoderma harzianum at 10-20 microM. The results of confocal laser scanning microscopy and SYTOX Green uptake demonstrated that its effective region was the membrane of the fungal cell surface. In addition, this protein was found to be noncytotoxic and heat-stable. Taken together, the results of this study indicate that Pr-2 is a good candidate for use as a natural antifungal agent.

New stilbenoid with inhibitory activity on viral neuraminidases from Erythrina addisoniae.

Influenza occurs with seasonal variations and reaches the peak prevalence in winter causing the death of many people worldwide. A few inhibitors of viral neuraminidase, including amantadine, rimantadine, zanamivir, and oseltamivir, have been used as influenza therapy. However, as drug-resistant influenza viruses are generated rapidly, there is a need to identify new agents for chemotherapy against influenza. Therefore, research on more effective drugs has been given high priority. During the course of an anti-influenza screening program on natural products, two new compounds (1 and 2) along with seven known flavonoid derivatives (3-9) were isolated as active principles from an EtOAc-soluble extract of the root bark of Erythrina addisoniae. The stilbenoid (2) and chalcone (3, 4, and 6) compounds of the isolates exhibited stronger activity than the isoflavone ones. Compound 2, which is a formylated stilbenoid derivative, exhibited strong inhibition of both influenza H1N1 and H9N2 neuraminidases with IC(50) values of 8.80±0.34 μg/mL and 7.19±0.40 μg/mL, respectively.

Analysis of Arabidopsis glucose insensitive growth Mutants Reveals the Involvement of the Plastidial Copper Transporter PAA1 in Glucose-Induced Intracellular Signaling

Sugars play important roles in many aspects of plant growth and development, acting as both energy sources and signaling molecules. With the successful use of genetic approaches, the molecular components involved in sugar signaling have been identified and their regulatory roles in the pathways have been elucidated. Here, we describe novel mutants of Arabidopsis (Arabidopsis thaliana), named glucose insensitive growth (gig), identified by their insensitivity to high-glucose (Glc)-induced growth inhibition. The gig mutant displayed retarded growth under normal growth conditions and also showed alterations in the expression of Glc-responsive genes under high-Glc conditions. Our molecular identification reveals that GIG encodes the plastidial copper (Cu) transporter PAA1 (for P1B-type ATPase 1). Interestingly, double mutant analysis indicated that in high Glc, gig is epistatic to both hexokinase1 (hxk1) and aba insensitive4 (abi4), major regulators in sugar and retrograde signaling. Under high-Glc conditions, the addition of Cu had no effect on the recovery of gig/paa1 to the wild type, whereas exogenous Cu feeding could suppress its phenotype under normal growth conditions. The expression of GIG/PAA1 was also altered by mutations in the nuclear factors HXK1, ABI3, and ABI4 in high Glc. Furthermore, a transient expression assay revealed the interaction between ABI4 and the GIG/PAA1 promoter, suggesting that ABI4 actively regulates the transcription of GIG/PAA1, likely binding to the CCAC/ACGT core element of the GIG/PAA1 promoter. Our findings indicate that the plastidial Cu transporter PAA1, which is essential for plastid function and/or activity, plays an important role in bidirectional communication between the plastid and the nucleus in high Glc.

Over-expression of the IGI1 leading to altered shoot-branching development related to MAX pathway in Arabidopsis

Shoot branching and growth are controlled by phytohormones such as auxin and other components in Arabidopsis. We identified a mutant (igi1) showing decreased height and bunchy branching patterns. The phenotypes reverted to the wild type in response to RNA interference with the IGI1 gene. Histochemical analysis by GUS assay revealed tissue-specific gene expression in the anther and showed that the expression levels of the IGI1 gene in apical parts, including flowers, were higher than in other parts of the plants. The auxin biosynthesis component gene, CYP79B2, was up-regulated in igi1 mutants and the IGI1 gene was down-regulated by IAA treatment. These results indicated that there is an interplay regulation between IGI1 and phytohormone auxin. Moreover, the expression of the auxin-related shoot branching regulation genes, MAX3 and MAX4, was down-regulated in igi1 mutants. Taken together, these results indicate that the overexpression of the IGI1 influenced MAX pathway in the shoot branching regulation.

Isolation and Purification of a Novel Deca-Antifungal Peptide from Potato (Solanum tuberosum L. cv. Jopung) Against Candida albicans

In a previous study, an antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) and by gel filtration and HPLC. In this study, the functional peptide was characterized by partial acid digestion using HCl and HPLC. We obtained three peaks from the AFP-J, the first and third peaks were not active in the tested fungal strain. However, the second peak, which was named Potide-J, was active (MIC; 6.25 μg/mL) against Candida albicans. The amino acid sequences were analyzed by automated Edman degradation, and the amino acid sequence of Potide-J was determined to be Ala-Val-Cys-Glu-Asn-Asp-Leu-Asn-Cys-Cys. Mass spectrometry showed that its molecular mass was 1083.1 Da. Finally, we confirmed that a disulfide bond was present between Cys3 and Cys9 or Cys10. Using this structure, Potide-J was synthesized via solid-phase methods. In these experiments, only the linear sequence was shown to display strong activity against Candida albicans. These results suggest that Potide-J may be an excellent candidate compound for the development of commercially applicable antibiotic agents.

Isolation and Characterization of an Extracellular Antimicrobial Protein from Aspergillus oryzae

A 17 kDa antimicrobial protein was isolated from growth medium containing the filamentous fungus Aspergillus oryzae by extracting the supernatants from the culture media, ion exchange chromatography on CM-sepharose, and C18 reverse-phase high-performance liquid chromatography. This antimicrobial protein, which we considered to be an extracellular antimicrobial protein from A. oryzae (exAP-AO17), possessed antimicrobial activity but lacked hemolytic activity. The exAP-AO17 protein strongly inhibited pathogenic microbial strains, including pathogenic fungi, Fusarium moniliform var. subglutinans and Colletotrichum coccodes, and showed antibacterial activity against bacteria, including E. coli O157 and Staphylococcus aureus. To confirm that the protein acts as a regulation factor for extracellular secretion, we examined growth under varying conditions of N sources, C sources, ions, ambient pH, and stress. Various culture conditions were found to induce characteristic changes in the expression of protein synthesis as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Highly basic polypeptides were regulated by suppressing the ambient pH under acidic conditions and strongly induced under alkaline conditions, thus confirming that pH regulation is physiologically relevant. The expression of exAP-AO17 was upregulated by heat shock upon growth in the presence of NaCl. Automated Edman degradation showed that the N-terminal sequence of exAP-AO17 was NH 2-GLPGPAGAVGFAGKDQNM-. ExAP-AO17 showed partial sequence homology with a collagen belonging to the animal source. These results suggest that exAP-AO17 is an excellent candidate as a lead compound for the development of novel oral or other types of anti-infective agents.