Gung-Pyo Lee

Cloning and Phylogenetic Characterization of Coat Protein Genes of Two Isolates of Apple mosaic virus from ¡？Fuji¡？ Apple

Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was detected and isolated from diseased `Fuji` apple (Malus domestica) in Korea. The coat protein (CP) genes of two ApMV strains, denoted as ApMV-Kl and ApMV-K2, were amplified by using the reverse transcription and polymerase chain reaction (RT-PCR) and were analyzed thereafter. The objectives were to define the molecular variability of genomic information of ApMV found in Korea and to develop virus-derived resistant gene source for making virus-resistant trans-genic apple. RT-PCR amplicons for the APMVS were cloned and their nucleotide sequences were determined. The CPs of ApMV-Kl and ApMV-K2 consisted of 222 and 232 amino acid residues, respectively. The identities of the CPs of the two Korean APMVS were 93.1% and 85.6% at the nucleotide and amino acid sequences, respectively. The CP of ApMV-Kl showed 46.1-100% and 43.2-100% identities to eight different ApMV strains at the nucleotide and amino acid levels, respectively. When ApMV-PV32 strain was not included in the analysis, ApMV strains shared over 83.0% and 78.6% homologies at the nucleotide and amino acid levels, respectively. ApMV strains showed heterogeneity in CP size and sequence variability. Most of the amino acid residue differences were located at the N-termini of the strains of ApMV, whereas, the middle regions and C-termini were remarkably conserved. The APMVS were 17.(1-54.5% identical with three other species of the genus Ilarviyus. ApMV strains can be classified into three subgroups (subgroups I, II, and III) based on the phylogenetic analysis of CP gene in both nucleotide and amino acid levels. Interestingly, all the strains of subgroup I were isolated from apple plants, while the strains of subgroups II and III were originated from peach, hop, or pear, The results suggest that ApMV strains co-evolved with their host plants, which may have resulted in the CP heterogeneity.

Alteration of floral organ identity by over-expression of IbMADS3-1 in tobacco

The MADS-box genes have been studied mainly in flower development by researching flower homeotic mutants. Most of the MADS-box genes isolated from plants are expressed exclusively in floral tissues, and some of their transcripts have been found in various vegetative tissues. The genes in the STMADS subfamily are important in the development of whole plants including roots, stems, leaves, and the plant vascular system. IbMADS3-1, which is in the STMADS subfamily, and which has been cloned in Ipomoea batatas (L.) Lam., is expressed in all vegetative tissues of the plant, particularly in white fibrous roots. Sequence similarity, besides the spatial and temporal expression patterns, enabled the definition of a novel MADS-box subfamily comprising STMADS16 and the other MADS-box genes in STMADS subfamily expressed specifically in vegetative tissues. Expression of IbMADS3-1 was manifest by the appearance of chlorophyll-containing petals and production of characteristic changes in organ identity carpel structure alterations and sepaloidy of the petals. In reverse transcription-polymerase chain reaction analysis with a number of genes known to be key regulators of floral organ development, the flowering promoter NFL1 was clearly reduced at the RNA level compared with wild type in transgenic line backgrounds. Moreover, NtMADS5 showed slight down-regulation compared with wild-type plants in transgenic lines. These results suggest that IbMADS3-1 could be a repressor of NFL1 located upstream of NtMADS5. IbMADS3-1 ectopic expression is suggested as a possible means during vegetative development by which the IbMADS3-1 gene may interfere with the floral developmental pathway.

Improvement of floral scent of ornamentals via metabolic engineering

Abstract Floral scent emitted from many plants is the key factor for pollinator attraction and defense for survival in nature and is important industrial materials for perfumery as well. It is a complex mixture of various organic molecules with a high volatility or a high vapor pressure. In general, floral scents are divided into three categories, aliphatics, terpenoids, and phenylpropanoids/benzenoids, based on its origin. About 1,700 scent compounds have been identified and their biochemistry and molecular biology also have elucidated their biosynthesis from various flowering plants during the last ten years. In addition to improvement of vase life, flower color and shape, and/or disease resistance, floral scent is coming up to the major breeding target for improve-ment of marketability. Therefore, metabolic engineering can be an important tool in near future and may be able to facilitate the breeding program for novel cultivar selection and improvement of marketability of floricultural crops.

Characterization of an Isolate of Cucumber mosaic virus Isolated from Chinese aster (Callistephus chinensis)

An isolate of Cucumber mosaic virus (CMV), designated as Cas-CMV, was isolated from Chinese aster (Callistephus chinensis) showing severe mosaic symptom, and its properties was compared to the well-characterized Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, and restriction enzyme profile of the PCR products. Cas-CMV differed markedly in their host reaction to Fny-CMV or As-CMV in Cucurbita pepo cv. Black beauty. In the zucchini squash, all strains induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves. However, symptoms induced by Cas-CMV were developed lethal necrosis on the young plants 15 to 20 days after inoculation. In experiments of dsRNA analysis and RT-PCR analysis, properties of Cas-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using HindIII of the RT-PCR products showed that Cas-CMV belong to a member of CMV subgroup IA.

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca2+-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.

Characterization and expression pattern of IbPRP1 and IbPRP2 stress-related genes from sweetpotato

Two putative stress-related genes were isolated from sweet-potato and were designated as IbPRP1 and IbPRP2 (Ipomoea batatas proline-rich proteins 1 and 2). The deduced amino acid aligment of IbPRP1 and IbPRP2 shows that these two genes belong to the AAI_LTSS superfamily. Proteins in this family are known to play primary roles including defending plants from pathogens and insects, lipid transport between intracellular membranes, and nutrient storage. The mRNA expression of IbPRP1 and IbPRP2 were investigated and the results demonstrate that IbPRP2 has tissue-specific expression. Moreover, IbPRP1 and IbPRP2 may be involved in response to various stresses including drought, pathogen, and oxidative stress. In addition, when leaf disc test was performed, the IbPRP1 transgenic tobacco plants showed increase in tolerance to salt (100 mM, 200 mM, and 300 mM). Moreover, IbPRP1 and IbPRP2 may have some roles of transmembrane protein in sweetpotato when checked through GFP fusion cell localization and transmembrane analysis. All of these results indicate that IbPRP1 and IbPRP2 might play an important role in plant stress responses.

In vitro plant regeneration from axillary buds of Hibiscus syriacus L.

Presently, we report a simple, reproducible and high frequency plant regeneration in Hibiscus syriacus L. using axillary buds. H. syriacus was regenerated from axillary buds directly or through a callus phase. Regenerated shoots were directly induced from young and fresh axillary buds cultured on Murashige and Skoog medium (MS) supplemented with 0.01 mg/L of the growth regulator thidiazuron (TDZ) after 2 weeks of culture. Directly induced shoots were transferred to hormone-free MS medium and root development was observed after 6 weeks. On the other hand, old and stale axillary buds were regenerated to shoots via callus induction on MS medium containing 0.01–2 mg/L TDZ after 4 weeks. A TDZ concentration of 0.01 mg/L was most effective in callus formation. Green callus was transferred to MS medium containing 0.01 mg/L α-naphthalene acetic acid (NAA) and 0.5 mg/L benzylaminopurine (BA). After 4 weeks, callus had developed into multiple shoots. Plantlets were formed from 10 week cultures of single shoots on hormone-free MS medium. Regenerated plantlets were cultured on MS medium for one month and then transferred to pots containing garden soil. Potted plants were acclimatized for one month and grown to maturity under greenhouse conditions. The present study has shown that various concentrations of plant growth regulator can be effective for in vitro plant regeneration of H. syriacus. The direct and indirect regeneration protocol presented here will be useful for understanding the manipulation and propagation of H. syriacus.

Pollen allergic risk assessment of genetically modified virus resistant pepper and functional Chinese cabbage

Pollen of genetically modified (GM) pepper containing the gene for cucumber mosaic virus (CMV) coat protein (CP) and GM Chinese cabbage with high phenylethylisothiocyanate (PEITC) content was investigated for assessment of allergic risk. Amino acid (AA) sequences of the inserted gene products of GM virus resistant pepper and GM Chinese cabbage with high PEITC content were compared with those of known allergens. No known allergen greater than 35% AA sequence homology, over 80 AA window or more than 8 consecutive identical AA was found. Protein patterns of GM/non-GM pepper and Chinese cabbage pollen extracts in SDS-PAGE analysis showed the same distribution of protein bands among the GM and non-GM pepper or Chinese cabbage, respectively. Sera from pollen allergic patients showed some IgE reactivity via immunoblotting and ELISA; however, no differences were observed between the pollen of GM and non-GM pepper or Chinese cabbage, respectively. Based on these results, we conclude that pollens of the virus resistant GM pepper and GM Chinese cabbage with high PEITC have no differences in their protein composition or allergenicity relative to non-GM pepper and Chinese cabbage.

Effect of reactive oxygen species on floral senescence in Hibiscus syriacus L.

To understand the effect of reactive oxygen species (ROS) on floral senescence in Hibiscus syriacus L., we have investigated change in relative water potential, malondialdehyde (MDA) content, H2O2 content and the activity of antioxidative enzymes in the petals during flower opening and senescence. Hibiscus flowers were achieved full bloom at early morning and started to in-rolling and showed petal in-rolling over than 50% at 24 h and 36 h after full bloom, respectively. The flower was a decrease in fresh weight by 30% and showed water loss with floral senescence. MDA content and activity of antioxidative enzymes such as APX, GR and CAT were showed no significant change until 36 h after full bloom. In the flower 48 h after full bloom that showed complete petal in-rolling and wilting, however, activity of antioxidative enzymes and H2O2 content was greatly increased as compared with 0 h after full bloom. These results suggest that reactive oxygen species are related to accelerating the later senescence more than inducing the early senescence during Hibiscus flower senescence.

Transformation of Fuji Apple Plant Harboring the Coat Protein Gene of Cucumber mosaic virus

Transformation of Fuji apple (Malus domestica `Fuji`) was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.