Si-Yong Kang

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.

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.

Analysis of the genetic relationship of gamma-irradiated in vitro mutants derived from standard-type chrysanthemum cv. Migok

In Korea, chrysanthemum is the second-most popular cutflower next to roses and can be divided into two main groups: spray and standard types according to their usage. The standard-type chrysanthemums are often used for condolence, and the important traits of this type include lack of branching and a long flowering period. This study was performed to compare the polymorphisms, genetic diversity, and genetic distances between the original variety and gamma-irradiated in vitro plants using amplified fragment length polymorphism (AFLP) markers. The in vitro explants derived from a standard-type flower, ‘Migok’, were gamma-irradiated at 0, 30, 50, 70, and 100 Gy, and the resulting genetic variations were more diverse in the in vitro populations than in the plants derived from conventional cuttings. We identified 83% (866 bands) of the polymorphisms using 12 primer combinations, and the highest polymorphism was detected using the M-CAT/E-ACC combination with a 30 Gy irradiation treatment. In addition, the genetic diversity and polymorphic information content value were the highest at 30 Gy irradiation of the in vitro population, whereas the genetic distance was the furthest between the 30 Gy-irradiated and other populations. Therefore, 30 Gy is the most effective dosage for inducing in vitro genetic variations in standard-type Migok.

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.

Selection and molecular characterization of a high tocopherol accumulation rice mutant line induced by gamma irradiation

Tocopherols are micronutrients with antioxidant properties. They are synthesized by photosynthetic bacteria and plants, and play important roles in animal and human nutrition. In this study, we isolated a new rice mutant line with elevated tocopherol content (MRXII) from an in vitro mutagenized population induced by gamma irradiation. The mutant exhibited greater seed longevity than the control, indicating a crucial role for tocopherols in maintaining viability during quiescence, and displayed faster seedling growth during the early growth stage. To study the molecular mechanism underlying vitamin E biosynthesis, we examined the expression patterns of seven rice genes encoding vitamin E biosynthetic enzymes. Accumulation levels of the OsVTE2 transcript and OsVTE2 protein in the MRXII mutant were significantly higher than in the control. Sequence analysis revealed that the MRXII mutant harbored a point mutation in the OsVTE2 promoter region, which resulted in the generation of MYB transcription factor—binding cis-element. These results help identify the promoter regions that regulate OsVTE2 transcription, and offer insights into the regulation of tocopherol content.

Comparative gene expression analysis in a highly anthocyanin pigmented mutant of colorless chrysanthemum.

In this study, we investigated differentially expressed genes between the original chrysanthemum cultivar ‘Argus’ with white flower color and its gamma-ray irradiated mutant ‘ARTI-purple’ with purple flower color. The expression levels of anthocyanin biosynthetic genes were not associated with anthocyanin accumulations of Argus and ARTI-purple. Expressed sequence tags (ESTs) analysis was performed to identify a novel cDNAs encoding enzymes of specific plant metabolic pathways and the biological effects of gamma-ray mutation through alterations in expression in each flower. A total of 796 unigenes were isolated from chrysanthemum ray florets. These unigenes were functionally classified using gene ontologies and tentative pathway associations were established to 99 sequences in the Kyoto encyclopedia of genes and genomes. The expressions of the isolated ESTs were screened by cDNA dot blot hybridization. Seven differentially expressed genes were identified as being involved in carbohydrate and lipid metabolic pathways and five as transcription factor or signal transduction genes. Of particular note, decreased expression of CmMYB1 was identified at the ‘ARTI-purple’. The CmMYB1 shared high similarity with AtMYB4 and AtMYBL2 which is a negative regulator of anthocyanin and flavonol accumulation. Furthermore, two genes involved in lipid metabolism, enoyl-ACP reductase and [acyl-carrier-protein] S-malonyltransferase, were decreased in the ‘ARTI-purple’ flower. Our results suggest that the purple pigmentation of the ‘ARTI-purple’ is not just dependent on the expression of anthocyanin synthesis genes, and that the pigmentation may also affect other metabolic processing and the plant cell environment.

Analysis of genetic similarity detected by AFLP and PCoA among genotypes of kenaf (Hibiscus cannabinus L.).

Seventeen kenaf varieties collected from several regions around Asia and Europe were grown in Korea and their genetic diversity was analyzed using morphological characters and AFLP technique. In the morphological analysis, the 17 varieties were divided into two major groups according to stem diameter, plant height, and flowering periods. The late varieties, which could yield more biomass compared with the early-medium varieties, were included in one of two major groups. Nonetheless, it is difficult to identify individual varieties based on morphological characters because of their limited variation. For the AFLP analysis, 34 primer combinations generated a total of 3,193 polymorphic bands (out of 3,914) with a polymorphic rate of 82%. The clusters were divided into two major groups with a similarity coefficient of 0.63 by UPGMA analysis method; but each group did not show a common tendency. Additionally, the results of the AFLP analysis did not show similar tendency compared with morphological data, a result that might be explained in terms of convergent evolution, i.e. the acquisition of morphologically similar traits between distinctly unrelated varieties.

The identification of candidate radio marker genes using a coexpression network analysis in gamma-irradiated rice.

Plant physiological and biochemical processes are significantly affected by gamma irradiation stress. In addition, gamma-ray (GA) differentially affects gene expression across the whole genome. In this study, we identified radio marker genes (RMGs) responding only to GA stress compared with six abiotic stresses (chilling, cold, anoxia, heat, drought and salt) in rice. To analyze the expression patterns of differentially expressed genes (DEGs) in gamma-irradiated rice plants against six abiotic stresses, we conducted a hierarchical clustering analysis by using a complete linkage algorithm. The up- and downregulated DEGs were observed against six abiotic stresses in three and four clusters among a total of 31 clusters, respectively. The common gene ontology functions of upregulated DEGs in clusters 9 and 19 are associated with oxidative stress. In a Pearsons correlation coefficient analysis, GA stress showed highly negative correlation with salt stress. On the basis of specific data about the upregulated DEGs, we identified the 40 candidate RMGs that are induced by gamma irradiation. These candidate RMGs, except two genes, were more highly induced in rice roots than in other tissues. In addition, we obtained other 38 root-induced genes by using a coexpression network analysis of the specific upregulated candidate RMGs in an ARACNE algorithm. Among these genes, we selected 16 RMGs and 11 genes coexpressed with three RMGs to validate coexpression network results. RT-PCR assay confirmed that these genes were highly upregulated in GA treatment. All 76 genes (38 root-induced genes and 38 candidate RMGs) might be useful for the detection of GA sensitivity in rice roots.

Variation in the phenotypic features and transcripts of color mutants of chrysanthemum (Dendranthema grandiflorum) derived from gamma ray mutagenesis

We investigated the structural genes and their transcripts for anthocyanin synthesis inDendranthema grandiflorum ‘Argus’. Color variations in chrysanthemum mutants were obtained through gamm ray irradiation to regenerated plants from anin vitro. Normal florets were pinkish, but the mutants had white or purple ray florets and white, purple, or yellow-green disc florets. Irradiation modified both flower size and the number of ray florets. Compared with the control, levels of total anthocyanins in the mutants ranged from 4 times lower to 6 times higher for the disc florets. This disparity was even more evident, up to 14-fold greater, in the ray florets. Expression of the CHI, F3′H, F3′5′H, DFR, and LDOX genes varied among the mutants, but no dramatic changes were detected in CHS and F3H transcripts in either leaf or floret tissues. Sequence homology to known anthocyanin genes from other plant species was 61 to 84%, 62 to 74%, and 71 to 76% for CHI, F3′H, and LDOX, respectively. Our results support the proposal that such radiation-induced mutations in genes within the anthocyanin pathway are associated with variations in chrysanthemum flower color.