Jae-Wook Bang

Overexpression of sweetpotato swpa4 peroxidase results in increased hydrogen peroxide production and enhances stress tolerance in tobacco

Plant peroxidases (POD) reduce hydrogen peroxide (H2O2) in the presence of an electron donor. Extracellular POD can also induce H2O2 production and may perform a significant function in responses to environmental stresses via the regulation of H2O2 in plants. We previously described the isolation of 10 POD cDNA clones from cell cultures of sweetpotato (Ipomoea batatas). Among them, the expression of the swpa4 gene was profoundly induced by a variety of abiotic stresses and pathogenic infections (Park et al. in Mol Gen Genome 269:542–552 2003; Jang et al. in Plant Physiol Biochem 42:451–455 2004). In the present study, transgenic tobacco (Nicotiana tabacum) plants overexpressing the swpa4 gene under the control of the CaMV 35S promoter were generated in order to assess the function of swpa4in planta. The transgenic plants exhibited an approximately 50-fold higher POD specific activity than was observed in control plants. Both transient expression analysis with the swpa4-GFP fusion protein and POD activity assays in the apoplastic washing fluid revealed that the swpa4 protein is secreted into the apoplastic space. In addition, a significantly enhanced tolerance to a variety of abiotic and biotic stresses occurred in the transgenic plants. These plants harbored increased lignin and phenolic content, and H2O2 was also generated under normal conditions. Furthermore, they showed an increased expression level of a variety of apoplastic acidic pathogenesis-related (PR) genes following enhanced H2O2 production. These results suggest that the expression of swpa4 in the apoplastic space may function as a positive defense signal in the H2O2-regulated stress response signaling pathway.

Sweetpotato late embryogenesis abundant 14 ( IbLEA14 ) gene influences lignification and increases osmotic- and salt stress-tolerance of transgenic calli

Late embryogenesis abundant 14 (LEA14) cDNA was isolated from an EST library prepared from dehydration-treated fibrous roots of sweetpotato (Ipomoea batatas). Quantitative RT-PCR revealed a variety of different IbLEA14 expression patterns under various abiotic stress conditions. IbLEA14 expression was strongly induced by dehydration, NaCl and abscisic acid treatments in sweetpotato plants. Transgenic sweetpotato non-embryogenic calli harboring IbLEA14 overexpression or RNAi vectors under the control of CaMV 35S promoter were generated. Transgenic calli overexpressing IbLEA14 showed enhanced tolerance to drought and salt stress, whereas RNAi calli exhibited increased stress sensitivity. Under normal culture conditions, lignin contents increased in IbLEA14-overexpressing calli because of the increased expression of a variety of monolignol biosynthesis-related genes. Stress treatments elicited higher expression levels of the gene encoding cinnamyl alcohol dehydrogenase in IbLEA14-overexpressing lines than in control or RNAi lines. These results suggest that IbLEA14 might positively regulate the response to various stresses by enhancing lignification.

Suppression of RICE TELOMERE BINDING PROTEIN1 Results in Severe and Gradual Developmental Defects Accompanied by Genome Instability in Rice

Although several potential telomere binding proteins have been identified in higher plants, their in vivo functions are still unknown at the plant level. Both knockout and antisense mutants of RICE TELOMERE BINDING PROTEIN1 (RTBP1) exhibited markedly longer telomeres relative to those of the wild type, indicating that the amount of functional RTBP1 is inversely correlated with telomere length. rtbp1 plants displayed progressive and severe developmental abnormalities in both germination and postgermination growth of vegetative organs over four generations (G1 to G4). Reproductive organ formation, including panicles, stamens, and spikelets, was also gradually and severely impaired in G1 to G4 mutants. Up to 11.4, 17.2, and 26.7% of anaphases in G2, G3, and G4 mutant pollen mother cells, respectively, exhibited one or more chromosomal fusions, and this progressively increasing aberrant morphology was correlated with an increased frequency of anaphase bridges containing telomeric repeat DNA. Furthermore, 35S:anti-RTBP1 plants expressing lower levels of RTBP1 mRNA exhibited developmental phenotypes intermediate between the wild type and mutants in all aspects examined, including telomere length, vegetative and reproductive growth, and degree of genomic anomaly. These results suggest that RTBP1 plays dual roles in rice (Oryza sativa), as both a negative regulator of telomere length and one of positive and functional components for proper architecture of telomeres.

Integration of Cytogenetic and Genetic Linkage Maps Unveils the Physical Architecture of Tomato Chromosome 2

We report the integration of the linkage map of tomato chromosome 2 with a high-density bacterial artificial chromosome fluorescence in situ hybridization (BAC–FISH)-based cytogenetic map. The euchromatic block of chromosome 2 resides between 13 and 142 cM and has a physical length of 48.12 μm, with 1 μm equivalent to 540 kb. BAC–FISH resolved a pair of loci that were 3.7–3.9 Mb apart and were not resolved on the linkage map. Most of the regions had crossover densities close to the mean of ∼200 kb/cM. Relatively hot and cold spots of recombination were unevenly distributed along the chromosome. The distribution of centimorgan/micrometer values was similar to the previously reported recombination nodule distribution along the pachytene chromosome. FISH-based physical maps will play an important role in advanced genomics research for tomato, including map-based cloning of agronomically important traits and whole-genome sequencing.

Molecular cytogenetic mapping of Cucumis sativus and C. melo using highly repetitive DNA sequences

Chromosomes often serve as one of the most important molecular aspects of studying the evolution of species. Indeed, most of the crucial mutations that led to differentiation of species during the evolution have occurred at the chromosomal level. Furthermore, the analysis of pachytene chromosomes appears to be an invaluable tool for the study of evolution due to its effectiveness in chromosome identification and precise physical gene mapping. By applying fluorescence in situ hybridization of 45S rDNA and CsCent1 probes to cucumber pachytene chromosomes, here, we demonstrate that cucumber chromosomes 1 and 2 may have evolved from fusions of ancestral karyotype with chromosome number n = 12. This conclusion is further supported by the centromeric sequence similarity between cucumber and melon, which suggests that these sequences evolved from a common ancestor. It may be after or during speciation that these sequences were specifically amplified, after which they diverged and specific sequence variants were homogenized. Additionally, a structural change on the centromeric region of cucumber chromosome 4 was revealed by fiber-FISH using the mitochondrial-related repetitive sequences, BAC-E38 and CsCent1. These showed the former sequences being integrated into the latter in multiple regions. The data presented here are useful resources for comparative genomics and cytogenetics of Cucumis and, in particular, the ongoing genome sequencing project of cucumber.

Differential expression of four sweet potato peroxidase genes in response to abscisic acid and ethephon

Expression of four peroxidase (POD) genes, three anionic PODs (swpa1, swpa2 and swpa3), and one neutral POD (swpn1) isolated from suspension cultures of sweet potato (Ipomoea batatas) were analyzed by measuring the accumulation of transcripts in suspension cultured cells and leaves of sweet potato in response to the stress-related plant hormones abscisic acid (ABA) and ethephon (an ethylene generating chemical). The four genes responded differently to ABA (0.1 mM) and ethephon (0.1 mM) in cultured cells and leaves. In suspension cultures, ABA reduced the expression levels of swpa1, swpa2, and swpn1, but did not affect the level of swpa3. Ethephon strongly increased expression levels of swpa3 and swpn1, and slightly increased the level of swpa1. The expression level of swpa2 was reduced. Expression levels in intact leaves, however, were significantly changed by this treatment. Expression of the swpa1 and swpa2 genes was induced 15 min after ABA treatment, followed by a decrease to a basal level after 3 h. A strong re-expression occurred after 12 h. Expression of the swpa3 and swpn1 genes occurred from 3 to 24 h after treatment. All four genes were differentially expressed 12 h after ethephon treatment. The swpa2 gene was strongly expressed immediately after ethephon treatment. The results indicate that each POD gene is differentially regulated by ABA and ethylene in whole plants and in cultured cells in vitro.

Physical Mapping of rRNA Gene Loci and Inter-specific Relationships in Wild Lilium Distributed in Korea

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1–7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber’s classification of the genus Lilium.

Rapid divergence of repetitive DNAs in Brassica relatives

Centromeric, subtelomeric, and telomeric repetitive DNAs were characterized in Brassica species and the related Raphanus sativus and Arabidopsis thaliana. In general, rapid divergence of the repeats was found. The centromeric tandem satellite repeats were differentially distributed in the species studied, suggesting that centromeric repeats have diverged during the evolution of the A/C and B genome lineages. Sequence analysis of centromeric repeats suggested rapid evolution. Pericentromere-associated retrotransposons were identified and showed divergence during the evolution of the lineages as centromeric repeats. A novel subtelomeric tandem repeat from B. nigra was found to be conserved across the diploid Brassica genomes; however, this sequence was not identified in the related species. In contrast to previous studies, interstitial telomere-like repeats were identified in the pericentromeres of Brassica chromosomes, and these repeats may be associated with genomic stability. These results provide insight into genome evolution during polyploidization in Brassica and divergence within the Brassicaceae.

Isolation and characterization of cDNA clones expressed under male sex expression conditions in a monoecious cucumber plant (Cucumis sativus L. cv. Winter Long)

In order to clone sex determination genes, both differential screening of a cDNA library and differential display reverse transcription PCR (DDRT-PCR) were conducted, using apex tissues from a Korean cucumber cultivar that exhibited different sex expression responses to differing photoperiods. Two genes (CsM1 and CsM2) expressed only under specific male expression conditions were then cloned via differential screening of a cDNA library and designated CsCYR (Cucumis sativus cyclin-related) and CsCYP (C. sativus cyclophilin). Both genes appear to be single-copy genes, and both express under male-expressing SD (short-day) conditions. CsM10 (C. sativus male specific clone 10) was isolated via DDRT-PCR, and was expressed preferentially under male expression conditions. The CsM10 gene encodes a non-coding RNA, and harbors a 179bp-conserved sequence, found in three genes which have been classified as a biotic stress signal-associated non-translatable RNA (or non-coding RNA) group. The CsM10 gene is also a single-copy gene, which is localized in the telomeric region of chromosome 6, and its expression is different in different tissues, developmental stages, and photoperiods. Based on the obtained sequence structures and expression patterns of CsM10 and its homologues, we suggest that CsM10 function appears to occur at the RNA level.

Physical mapping by FISH and GISH of rDNA loci and discrimination of genomes A and B inScilla scilloides complex distributed in Korea

The chromosomal locations of the 18S-26S (45S) and 5S rDNA loci in cytotypes AA, BB, and AABB ofScilla scilloides Complex from Korea were physically mapped using multicolor fluorescencein situ hybridization (McFISH). Genomicin situ hybridization (GISH) was also performed to distinguish between the AA and BB genomes in allotetraploid AABB plants. One 18S-26S rDNA locus was detected in both AA (a2) and BB (b1 ); one locus also was found in the allopolyploid AABB (b1 ). This demon-strated the loss of that locus in genome A. GISH with biotin-labeled DNA from the BB genome and digoxigenin-labeled 18S-26S rDNA probes revealed that the 18S-26S rDNA in AABB plants was localized in the nucleolus organizer region (NOR) of genome B. One and two 5S rDNA loci were found in diploids AA and BB, respectively. As expected, all three 5S rDNA loci were detected in the AABB plants. The sequence identities of the 5S rDNA genes among cytotypes AA and BB, AA and AABB, and BB and AABB were 99%, 95%, and 95%, respectively.