Young Myung Kwon

A novel extensin gene encoding a hydroxyproline-rich glycoprotein requires sucrose for its wound-inducible expression in transgenic plants.

A novel hydroxyproline-rich glycoprotein (SbHRGP3) that consists of two different domains is encoded by an extensin gene from soybean. The first domain (domain 1) located at the N terminus is composed of 11 repeats of Ser-Pro4-Lys-His-Ser-Pro4-Tyr3-His, whereas the second domain (domain 2) at the C terminus contains five repeats of Ser-Pro4-Val-Tyr-Lys-Tyr-Lys-Ser-Pro4-Tyr-Lys-Tyr-Pro-Ser-Pro5-Tyr-Lys-T yr- Pro-Ser-Pro4-Val-Tyr-Lys-Tyr-Lys. These two repeat motifs are organized in an extremely well-ordered pattern in each domain, which suggests that SbHRGP3 belongs to a new group of proteins having the repeat motifs of two distinct groups of dicot extensins. The expression of the SbHRGP3 gene increased with seedling maturation, and its expression was relatively high in the mature regions of the hypocotyl and in the root of soybean seedlings. An SbHRGP3-beta-glucuronidase (SbHRGP3-GUS) chimeric gene was constructed and expressed in transgenic tobacco plants. The expression of the SbHRGP3-GUS gene was not induced by wounding alone in transgenic tobacco plants; sucrose was also required. Expression was specific to phloem tissues and cambium cells of leaves and stems. In transgenic tobacco seedlings, SbHRGP3-GUS gene expression was activated by the maturation of the primary root and then inactivated; however, reactivation was specifically at the epidermis of the zone from which the lateral root was to be initiated. Its reactivation occurred just before the lateral root initiation. These results indicate that the SbHRGP3 gene in different tissues responds to different signals.

Molecular cloning and characterization of the gene encoding osmotin protein in Petunia hybrida

Abstract A cDNA clone encoding osmotin, PhOSM , was isolated from a cDNA library constructed from petal protoplast cultures of Petunia hybrida . PhOSM cDNA was composed of an open reading frame corresponding to protein of 246 amino acids and had a calculated molecular weight of 26.7 kDa. Database comparisons of the PhOSM protein sequences revealed high identity (89%) with the tobacco osmotin and tobacco OLP (Osmotin-Like-Protein). The increased accumulation of PhOSM mRNA in petal protoplast cultures was completely inhibited by treatment with Dhp (3,4-dehydro- l -proline), which is a inhibitor of peptydyl proline hydroxylation in cell wall regeneration. RNA blot analyses revealed that PhOSM was expressed primarily in roots and slightly in the pistil, 3 days after flowering. PhOSM expression was strongly induced in leaves that were exposed to Penicillium funiculosum , Erwinia stewartii and Pseudomonas syringae but not to Aspergillus nidulans . Upon wounding, PhOSM transcripts were induced in the directly damaged leaf but not systemically. Moreover, PhOSM transcript levels increased in response to octadecanoid pathway intermediates and treatments with aspirin and salicylic acid. Our results indicate that PhOSM is developmentally regulated as well as involved in wound-stress signal transduction.

Characterization of a cDNA encoding a proline-rich 14 kDa protein in developing cortical cells of the roots of bean (Phaseolus vulgaris) seedlings

A cDNA clone, corresponding to mRNAs preferentially expressed in the roots of bean (Phaseolus vulgaris L.) seedlings, was isolated. This clone contains a 381 bp open reading frame encoding a polypeptide of 13.5 kDa, designated PVR5 (Phaseolus vulgaris root 5). The amino acid sequence of this clone is rich in proline (13.5%) and leucine (12.7%) and shares significant amino acid sequence homology with root-specific and proline-rich proteins from monocots (maize and rice), and proline-rich proteins from dicots (carrot, oilseed rape, and Madagascar periwinkle). The precise biological roles of these polypeptides are unknown. PVR5 mRNA accumulation is developmentally regulated within the root, with high levels at the root apex and declining levels at distances further from the root tip. In situ hybridization shows that PVR5 mRNA specifically accumulates in the cortical ground meristem in which maximal cell division occurs. Southern blot analysis suggests that genomic DNA corresponding to PVR5 cDNA is encoded by a single gene or a small gene family.

Two closely related cDNAs encoding actin-depolymerizing factors of petunia are mainly expressed in vegetative tissues.

Actin-depolymerizing factor (ADF) is one of the small actin-binding proteins that regulate actin dynamics in cells. We have isolated two cDNA clones, PhADF1 and PhADF2, encoding ADF from cDNA libraries constructed from petal protoplast cultures and flowers of Petunia hybrida. PhADF1 and PhADF2 encode polypeptides of 139 and 143 amino acids with a calculated molecular mass of 16.04 and 16.51kDa, respectively. Co-sedimentation assay showed that the recombinant PhADF1 protein produced in Escherichia coli binds to F-actin at pH7. 0 and preferentially depolymerizes it at pH8.0. Gene tree analysis indicates that the plant ADF family can be grouped into four classes, and PhADFs are included in class I. Southern blot analyses revealed that one or two copies of PhADF genes are present in petunia genome, and several other related isoforms also exist. Northern blot analyses indicated that PhADF1 and PhADF2 are closely related and abundantly expressed in every plant organ except pollen. In addition, they are highly accumulated in mature vegetative tissue (petal, leaf, and stem). Our results indicate that the transcription of petunia ADF genes is differentially regulated by developmental signals.

Cortical tissue-specific accumulation of the root-specific ns-LTP transcripts in the bean (Phaseolus vulgaris) seedlings

The characterization of a cDNA clone encoding non-specific lipid transfer protein (PvLTP, formerly named PVR3) in the roots of bean seedlings has been previously reported. In this study, we examined the temporal and spatial accumulation of PvLTP mRNA and the effect of the auxin naphthaleneacetic acid (NAA) on the accumulation of PvLTP mRNA during root development. In situ hybridization showed that accumulation of PvLTP mRNA is highly tissue-specific. Accumulation was detected in the cortical tissue, but not in other tissues of root, including the quiescent center and root cap. Within the cortical tissue, accumulation of PvLTP mRNA was developmentally regulated; accumulation of PvLTP mRNA was high in the cortical tissue of the proximal and ground meristem and declined as cortical tissue developed further. Since the appropriate distribution of auxin is an important factor responsible for the maintenance of root meristem organization. We examined effect of auxin on the accumulation of PvLTP mRNA in relation to the development of cortical tissue. In bean seedlings grown on medium supplemented with 5 μM NAA, morphological alternations, including radial root expansion and abnormal tissue organization in the root apical meristem, were observed. Only faint accumulation signals of PvLTP mRNA were observed in the cortical tissue of proximal meristem region, indicating that cortical tissue development was repressed by exogenous NAA. However, our results suggest that the change in accumulation of PvLTP mRNA is not direct regulatory effect but reflective effect of altered development of cortical tissue that was induced by exogenous NAA. The temporal and spatial accumulation of PvLTP mRNA indicates that PvLTP is a useful marker for the development of cortical tissue in the root tip in bean seedlings.

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

Canavanine metabolism in tissue cultures of Canavalia lineata

The greening of callus was achieved by modulating the mediums growth regulator concentrations under continuous light. Canavalia lineata (L.) DC. calluses formed chlorophyll when they were exposed to continuous light in the presence of benzylaminopurine and indole-3-acetic acid. Canavanine and canaline were detected in the green callus. But only canaline was detected in the white callus grown in the dark. Feedings of canaline to suspension cultures showed that the green suspended cells were capable of de novo biosynthesis of canavanine, but the white suspended cells were not. Exogeneously supplied canavanine was used to produce canaline and homoserine by the white suspended cells. Arginase activity was induced by the addition of arginine or canavanine to the medium, and canaline reductase activity was induced by the addition of canaline but not with ornithine in the white suspended cells.

Purification and characterization of ornithine carbamoyltransferase from the chloroplasts of Canavalia lineata leaves

Abstract Ornithine carbamoyltransferase (OCT) was characterized from the chloroplasts of Canavalia lineata leaves. The OCT was purified 419-fold with a yield of 7.9% by streptomycin sulfate precipitation, ammonium sulfate fractionation, Sephacryl S-300 gel filtration, hydroxylapatite, and reactive red dye chromatographies. The approximate molecular weight of OCT was 107 kD by measurement from Sephacryl S-300 gel filtration. The subunit molecular weight of the enzyme was 38 kD based on SDS-PAGE. These results suggest that the native enzyme is a trimer. The OCT of C. lineata used not only ornithine but also canaline as a substrate. As for the ornithine-dependent activity, the enzyme had a pH optimum at 8.5 and Michaelis constants of 2.4 mM for l -ornithine and 0.21 mM for carbamoyl phosphate (CP). As for the canaline-dependent activity, Michaelis constants for l -canaline and CP were 11 and 0.13 mM, respectively, at pH 8.0 (optimum pH of canaline-dependent activity). S-carbamoyl- l -cysteine and l -cysteine were very strong inhibitors for the enzyme activity. Inhibition by l -cysteine was shown to be competitive with respect to ornithine, but that was shown to be noncompetitive with respect to canaline. The ornithine-dependent activity was inhibited by 90% in the presence of 2 mM Cu2+ and the canaline-dependent activity was inhibited by 91 and 100% with 2 mM Cd2+ and 2 mM Hg2+, respectively.

Enzyme activities of canavanine metabolism in Canavalia lineata L. Callus

Summary When ornithine, citrulline, and argininosuccinic acid were added to the suspension cultures of Canavalia lineata callus, the arginine content was increased by 47%, 44%, and 75% in white callus, and 24%, 28 %, and 26 % in green callus, respectively. Feeding of canaline or ureidohomoserine increased canavanine content in green callus but not in white callus. However, when canavaninosuccinic acid was added to the medium, canavanine was measured in the white and green calli. The activities of ornithine carbamyitransferase, agininosuccinate synthetase, and argininosuccinate lyase were higher in the green callus than in the white callus. The activities of canavanine-dependent arginase and canaline reductase in the white and green calli were similar. Activity ratios (green/white) of ornithine carbamyitransferase, argininosuccinate synthetase, and argininosuccinate lyase were 1.45, 2.83, and 2.76 in canavanine accumulation and 1.35, 1.32, and 2.40 in arginine accumulation, respectively. The favorable Cascade effect of canavanine accumulation was observed in the green callus as compared with the white callus. Among the three enzymes, ureidohomoserine-dependent argininosuccinate synthetase activity of green callus was significantly higher than that of white callus. White callus had one peak of argininosuccinate synthetase activity (ASS-2) and green callus had two peaks of argininosuccinate synthetase activity (ASS-1 and ASS-2) from the DEAE-Sephacel column chromatography. Citrulline-dependent and ureidohomoserine-dependent catalytic activities of argininosuccinate synthetase were detected at the same peaks. It is concluded that the synthesis of canavanine is regulated through the coordination of ornithine carbamyitransferase, argininosuccinate synthetase, and argininosuccinate lyase.

Identification of an isoform of ornithine carbamoyltransferase that can effectively utilize canaline as a substrate from the leaves of Canavalia lineata.

An isoform of ornithine carbamoyltransferase that can utilize effectively canaline as a substrate (C-OCT) was identified from the leaves of Canavalia lineata (Thunb.) DC. The molecular mass of native C-OCT was 109 kDa by Sephacryl S-200 gel filtration and that of the subunit was 36 kDa by SDS-PAGE and immunoblotting using the antibody against kidney bean ornithine carbamoyltransferase (OCT). C-OCT has pH optimum at 8.0 for canaline-dependent OCT activity and Michaelis constants of 9.6 mM for canaline and 0.24 mM for carbamoyl phosphate. To some extent, the C-OCT also showed ornithine-dependent OCT activity and a pH optimum of 8.5; Michaelis constants for ornithine and carbamoyl phosphate were 0.21 and 0.086 mM, respectively. The enzyme exhibit V(max) for canaline-dependent activity 14-fold higher than that for ornithine-dependent activity and the ratio of canaline-dependent activity to ornithine-dependent activity was 66-fold higher than that of OCT of the same plant. It is likely that this enzyme plays an important role in the canavanine synthesis from the canavanine-containing plants.