Kyung Jun Lee

Genome-wide transcriptome profiling of ROS scavenging and signal transduction pathways in rice (Oryza sativa L.) in response to different types of ionizing radiation.

Ionizing radiation directly and indirectly affects gene expression within the plant genome. To access the antioxidant response of rice to different types of ionizing radiation, rice seeds were exposed to gamma-ray, cosmic-ray and ion beam radiation. Exposure to ionizing radiation dramatically decreased the shoot length in all plants but not the root length compared with a non-irradiated plant. Electron spin resonance, confirmed that the number of free radicals in cell was greatly increased by different types of ionizing radiation. The measurement of the MDA, chlorophyll, carotenoids contents and activity of antioxidant enzymes revealed that gamma-ray and cosmic-ray, but not ion beam, ionization deceased chlorophyll and carotenoids contents, while all three ionization treatments increased the activities of peroxidase, ascorbate peroxidase, and superoxide dismutase compared with the non-irradiated plants. Microarray analysis using Affymetrix GeneChip was used to establish the gene transcript profiles of rice genes regarding ROS scavenging and signal transduction pathways after ionization treatment. Many of the rice genes involved in ROS scavenging and signal transduction pathways showed induction or repression that had increased more than twofold after ionization treatment. In particular, genes associated with electron transport, such as NADPH oxidase-like and alternative oxidase, were often down-regulated by more than twofold in response to the ionization treatments. In our transcriptomic profile analysis, we confirmed that the expression of rice genes associated with ROS scavenging and signal transduction pathways was induced or repressed to different degrees by the different types of ionizing radiations, as in other environmental stresses.

Identification of Kunitz trypsin inhibitor mutations using SNAP markers in soybean mutant lines

The Kunitz trypsin inhibitor (KTi) in soybean has several polymorphic types that are controlled by multiple alleles, which behave in a co-dominant fashion. Of these, Tia and Tib, which differ by nine amino acids, are the predominant types. In order to develop a single nucleotide amplified polymorphism (SNAP) marker for the classification of the predominant KTi types, Tia and Tib, and evaluate KTi activities by differing KTi type total 451 soybean mutant lines (M12–M16 generation) were incorporated in this study. Among 451 soybean mutants, 144 and 13 mutant lines showed decreased and increased trypsin inhibitor activity when compared with the original cultivars, respectively. To identify the KTi type, we designed a SNAP marker. Among 451 mutant lines from 12 soybean cultivars and landraces, 8 mutant lines derived from cvs. Baekwoon, Paldal and Suwon115 showed a change in KTi type when compared with the original cultivars using the SNAP marker. Five mutant lines in Suwon115 changed from Tib to Tia, while two mutant lines derived from cv. Baekwoon and one mutant line derived from cv. Paldal were changed from Tia to Tib. These changes of KTi types were confirmed by sequencing of the KTi genes and non-denaturing polyacrylamide gel electrophoresis of the KTi proteins. To identify the effect of KTi activity based on the change in KTi type, we measured the KTi activity using the three cultivars and eight mutant lines that showed changes in KTi type. Two mutant lines (BW-1 and 7-2) derived from cv. Baekwoon and one mutant line (PD-5-10) from cv. Paldal that changed from Tia to Tib showed lower activity than the original cultivar. In cv. Suwon115, five mutant lines that changed from Tib to Tia showed higher activity than the original cultivar. These results indicate that the designed SNAP marker was capable of identifying the KTi type and that Tia activity was higher than Tib activity in soybean.

Metabolomics investigation of flavonoid synthesis in soybean leaves depending on the growth stage

Soybean (Glycine max L.) leaves have unique nutraceutical and pharmacological benefits, and have been widely used as a source of healthy and functional food stuffs in Korea. In this study, we investigated the phytochemical metabolomic changes of soybean leaves depending on growth stages (maturation period) assessed based on UPLC–QTOF–MS analysis. Principal component analysis was carried out to trace the metabolite profiles of the phytochemicals from the vegetable stage (1D) through the seven reproductive stages (R1–R7). On the loading plot, significant changes in the contents of metabolites were found during the growth, and eight flavonoid kaempferol glycosides (2, 3, 6, 8, and 10), daidzein (14), genistein (17), and coumestrol (19) were evaluated as growth markers among the 19 isolated metabolites. The kaempferol glycosides were increasingly synthesized from the 1D to the R6 stage but decreased rapidly at stages R7–R8. The extensively synthesized daidzein and genistein were shown during seed growth in the pod (R5–R6), while coumestrol was increased significantly at stages R7–R8 (maturity period). The synthetic pathway of the flavonoids could be elucidated based on the concentration of the individual metabolites. These results demonstrate that the metabolite production changed depending on the growth stage; a possible pathway could be deduced using metabolomic analysis to provide information regarding physiological characterization and optimal harvesting time for crops.

Transcriptome profiling in response to different types of ionizing radiation and identification of multiple radio marker genes in rice.

Ionizing radiation (IR) affects gene expression from plant genomes. To monitor the genome-wide transcriptional changes induced by three types of IR, we used the rice Affymetrix GeneChip microarray to identify genes that are up- or down-regulated by gamma rays (GAs), cosmic rays (CRs) and ion beams (IBs). The overall expression patterns in rice seedlings generated from seeds exposed to GAs and IBs were similar but differed for CRs exposure. Expression profiles of genes involved in metabolic pathways and cellular response were identified using MapMan analysis. This result revealed that IRs induced gene expression related to sucrose-starch metabolisms; sugar and starch accumulation was significantly increased in response to three types of IR in rice. In addition, we compared the genes commonly up- or down-regulated by exposure to three types of IR and identified 53 candidate radio marker genes (RMGs) that were differentially regulated by radiation exposure but not by other stresses. Among these genes, we selected six RMGs commonly applicable to different types of IR by specific coexpression networks using the algorithm for the reconstruction of accurate cellular networks (aracne) and confirmed the expression of these genes by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Our results provided insight into the mechanisms of the responses to different types of IR and identified multiple marker genes to predict sensitivity to three types of IR.

The improvement of ginsenoside accumulation in Panax ginseng as a result of γ-irradiation

In this study, gamma rays were used to irradiate embryogenic calli induced from cotyledon explants of Panax ginseng Meyer. After the embryogenic calli were irradiated, they were transferred to adventitious roots using an induction medium; next, mutated adventitious root (MAR) lines with a high frequency of adventitious root formations were selected. Two MAR lines (MAR 5-2 and MAR 5-9) from the calli treated with 50 Gy of gamma rays were cultured on an NH4NO3-free Murashige and Skoog medium with indole-3-butyric acid 3 mg/L. The expression of genes related to ginsenoside biosynthesis was analyzed using reverse transcription polymerase chain reaction with RNA prepared from native ginseng (NG), non-irradiated adventitious root (NAR) and 2 MAR lines. The expression of the squalene epoxidase and dammarenediol synthase genes was increased in the MAR 5-2 line, whereas the phytosterol synthase was increased in the MAR 5-9 line. The content and pattern of major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) were analyzed in the NG, NAR, and 2 MAR lines (MAR 5-2 and MAR 5-9) using TLC and HPLC. In the TLC analysis, the ginsenoside patterns in the NG, NAR, and 2 MAR lines were similar; in contrast, the MAR 5-9 line showed strong bands of primary ginsenosides. In the HPLC analysis, compared with the NG, one new type of ginsenoside was observed in the NAR and 2 MAR lines, and another new type of ginsenoside was observed in the 2 MAR lines irradiated with gamma rays. The ginsenoside content of the MAR 5-9 line was significantly greater in comparison to the NG.

Improvement of soybean through radiation-induced mutation breeding techniques in Korea

Mutation breeding techniques have been used to induce new genetic variations and improve agronomic traits in soybean. In Korea, the Korea Atomic Energy Research Institute (KAERI) has unique radiation facilities to induce plant mutations and has been conducting soybean mutation breeding programmes since the mid-1960s. Until now, the KAERI has developed five soybean mutant cultivars exhibiting early maturity, high yield and seed-coat colour change. In this paper, we review these five mutant cultivars in terms of how to successfully induce unique agronomic characteristics through mutation breeding programmes. A number of induced mutants exhibiting null lipoxygenase enzymes, altered protein patterns or Kunitz trypsin inhibitor activity could serve as genetic resources for the genetic analysis of target genes, and one mutant population has been developed for a reverse genetic study.

Identification of gamma ray irradiation-induced mutations in membrane transport genes in a rice population by TILLING

A high-salt environment represents environmental stress for most plants. Those that can grow and thrive in such an environment must have membrane transport systems that can respond effectively. Plant roots absorb Na+ from the soil, and the plant must maintain Na+ homeostasis to survive salt stress. A major mechanism by which salt-tolerant plants adapt to salt stress is through modulation of ion transport genes. We have subjected a population of rice plants to mutagenesis, and identified lines with both single-nucleotide polymorphisms (SNPs) in membrane transport genes and altered responses to salt stress. Primers labeled with FAM or HEX fluorescent dyes were designed for nine target genes encoding membrane transport proteins that are believed to regulate salt stress tolerance. A TILLING (Targeting Induced Local Lesions IN Genome) assay was performed on 2,961 M2 rice mutant lines using electrophoresis. After the TILLING assay, a total of 41 mutant lines containing SNPs in the target genes were identified and screened. The average number of mutations per gene was 1/492 kb in lines having SNPs, and the percentage of mutation sites per total sequence was 0.67. Among the 41 lines, nine had altered sequences in the exon region of the genes. Of these nine lines, seven were tolerant to salt stress after exposure to 170 mM NaCl for three weeks, while the other two lines were not more salt-tolerant than the control lines. Furthermore, five mutant lines containing SNPs in the coding region of OsAKT1, OsHKT6, OsNSCC2, OsHAK11 and OsSOS1 showed changed expression levels for each gene. We conclude that variation in membrane transport genes, such as expression levels and protein structures, may affect the rice plants tolerance to salt stress. These mutations represent traits that may be selected for large rice mutant populations, permitting efficient acquisition of salt-tolerant lines.

The complete chloroplast genome sequence of Glycyrrhiza lepidota (Nutt.) Pursh - An American wild licorice

The wild species are considered as primary and secondary genepools for the world’s most important food crops. Here, we sequenced the complete chloroplast (cp) genome sequence of an American wild licorice, Glycyrrhiza lepidota for the first time to investigate their phylogenetic relationship among inverted-repeat-lacking clade (IRLC) legumes. The total length of the chloroplast genome is 127,939 bp, with 34.2% overall GC content. The chloroplast genome harbors 110 known genes, including 76 protein-coding genes, four ribosomal RNA genes, and 30 tRNA genes. Similar to other closely related plastomes, rpl22 and rps16 are absent. A total of 464 cp microsatellites (cpSSRs) were analyzed in the G. lepidota. The majority of the SSRs in this cp genome are penta-nucleotides (61.6%). Locally collinear blocks (LCBs) identified between the Glycyrrhiza glabra and G. lepidota cp genomes were showed that they were well conserved with respect to gene organization and order. Moreover, the phylogenetic analysis indicates that G. lepidota is closely related to its confamilial counterparts than to the other taxa of the IRLC legumes.

Phytochemical distribution and antioxidant activities of Korean adzuki bean (Vigna angularis) landraces

Total polyphenol content (TPC), total phenolic acid content (TPA), and total flavonoid content (TFC) in 209 Korean adzuki bean landraces were determined by colorimetric methods. Antioxidant effects were evaluated with the DPPH, ABTS, ferric reducing antioxidant power (FRAP), reducing power (RP), and SOD assays. TPC, TPA, and TFC in the 209 Korean adzuki bean landraces ranged from 1.1 to 11.7 mg gallic acid equivalents/g, 0.37 to 5.03 mg caffeic acid equivalents/g, and 0.17 to 0.91 mg quercetin equivalents/g, respectively. Antioxidant activities as assessed by the DPPH, ABTS, FRAP, PR, and SOD assays showed wide variation, ranging from 12.2 to 86.3 (IC50), 0.85 to 5.25 mg ascorbic acid equivalents (ASC)/g, 0.41 to 5.44 mg ASC/g, 0.54 to 1.83 mg ASC/g, and 60.4 to 142.8 (IC50), respectively. Using the relative antioxidant capacity index (RACI), we found that the IT189394 had the highest antioxidant activity. In clustering analysis, 209 Korean adzuki bean landraces were classified into three clusters. Among them, cluster I contained 22 landraces with higher antioxidant activities, TPC, TFC, and TPA and smaller seed sizes than the other clusters. We anticipate that these results will provide useful information for the development of adzuki bean-based functional foods.

The complete chloroplast genome sequence of wild oat, Avena sterilis L. (Poaceae) and its phylogeny

Abstract Wild oat, Avena sterilis L. is a stout broad-leaved annual grass resembling cultivated oats in general appearance. In this study, we sequenced the complete chloroplast (cp) genome sequence of A. sterilis for the first time to investigate their phylogenetic relationship in the family Poaceae. The complete cp genome sequence is 135,887 bp in length with 38.5% overall GC content and exhibits a typical quadripartite structure comprising one pair of inverted repeats (21,603 bp) separated by a small single-copy region (12,575 bp) and a large single-copy region (80,106). The cp genome encodes 111 unique genes, 76 of which are protein-coding genes, four rRNA genes, 30 tRNA genes, and 18 duplicated genes in the inverted repeat region. The phylogenetic analysis indicated A. sterilis closely clustered with the cultivated oat, A. sativa L.