Ionizing radiation directly and indirectly affects gene expression within the plant genome. To access the physiological response of rice to different types of ionizing radiation, rice seeds were exposed to gamma-ray and ion beam radiation. Exposure to ionizing radiation dramatically decreased the shoot length compared with non-irradiated plants. Fluorescence-activated-cell-sorting (FACs) was used to measure DNA contents. There were significant correlations of dose-dependent between irradiated plant and non-irradiated plant. The radicals induced by the ionizing radiation in the plant could be observed by electron spin resonance (ESR). It was confirmed that the number of free radicals in cell was greatly increased all irradiated plants than non-irradiated plant. A significant positive correlation was shown between ionizing radiation dose and signal intensity. In order to determine the Genetic diversity, AFLP analysis was conducted with the irradiated plant and non-irradiated plant. Based on band patterns, the cluster analysis was conducted to evaluate the genetic variation by using the UPGMA (Unweighted Pair Grouping Method of Averages). Genetic diversity of irradiated plants by low dose ion beam was the closest non-irradiated plant and irradiated by high dose gamma-ray was the furthest from non-irradiated. We describe the detailed methods of ionizing irradiation and discuss its applications in genetic research as well as plant breeding.

Ionizing radiation affects gene expression from plant genomes. To monitor the genome-wide transcriptional changes induced by three types of ionizing radiation, we used the rice RNA sequencing to identify genes that are up- or down-regulated by gamma rays (GAs), proton (PRs) and ion beams (IBs). The Oryza sativa jacalin-like lectin domain containing proteins (OsJAC1) gene was highly induced by GAs, PRs and IBs. OsJAC1 was selected based on the expression patterns of a genome-wide dataset of RNA sequencing. Many jacalin-related lectin genes have been shown to be associated with disease resistance, biotic and abiotic stress signaling. Therefore, we studied its expression pattern in response to different abiotic stress and phytohormone treatments. The expression patterns of OsJAC1 under two different abiotic stress conditions (salt and heat stress) and phytohormones (salicylic acid and methyl jasmonate) were examined. The transcripts of OsJAC1 were significantly induced in response to abiotic stress conditions, including salt and heat treatments. In addition, it was induced in response to the salicylic acid and methyl jasmonate treatments, respectively. To investigate the sub-cellular localization of OsJAC1, the gene was expressed as a fusion protein tagged with GFP, in tobacco leaf epidermis and examined under confocal microscope. The OsJAC1 was clearly localized at the nucleus. These results provide critical insights into the molecular functions of the rice jacalin-like lectin domain containing proteins as receptors of external signals.

‘Tocomi-1’, a new japonica rice cultivar derived from a 200 Gy gamma ray irradiation with high tocopherol content and red pericarp. The local adaptability test of MRXII-1001-1 was carried out from 2012 to 2014 and it was named as ‘Tocomi-1’ in 2014. This variety is medium matured with heading date of August 12 in honam plain area of Korea. This variety is about 80 cm tall culm length and 106 spikelets per panicle. Its 1,000 grain-weight of rice seeds is 25.4 g. The yield potential of this variety is about 5.15 MT/ha in local adaptability test for three years. This variety exhibited greater seed longevity than the Donganbyeo, indicating a crucial role for tocopherols in maintaining viability during quiescence, and displayed faster seedling growth during the early growth stage. Tocopherol contents was 50% higher than the Donganbyeo. 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 ‘Tocomi-1’ were significantly higher than in the Donganbyeo. Sequence analysis revealed that the ‘Tocomi-1’ 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 in ‘Tocomi-1’.

To define whole genome-level of structural variation by ionization energies and radiation doses in plant, the seeds of Ilpum rice cultivar were acutely irradiated with gamma rays (100Gy, 200Gy, and 400Gy) and ion-beams (20Gy, 40Gy, and 80Gy), respectively. Six M1 rice plants were re-sequenced by Hi-Seq2500 with Ilpum cultivar as control. The average sequencing coverage of the individuals was 10.6X, and the average mapping rate to the rice reference genome (IRGSP-1.0) sequence was 96.95%. The individual plants were irradiated with gamma-400Gy and ion-50Gy had highest variation of SNP with 471,837 and 469,147, respectively. The number of insertion/deletion was 77,500 and 77,106, the synonymous and frame-shift were 7,859 and 7,763 in above two individuals. Although high genome variation shown between Ilpum cultivar and irradiated individuals, there were non-correlation between number of variation and radiation doses. However, five individuals, except ion-20Gy, showed 33 common variant blocks (CVBs) spanning 6 Mb in whole rice genome (1.6%). The CVBs were distributed on 12 rice chromosomes, Chromosome 6 had biggest CVB (5 blocks, 1.3Mb), whereas chromosome 9 had smallest CVB (0.01Mb). Total five hundred fifty one genes were in CVBs which can regard radiation sensitive genes or may be regarded as radiation hot spots in rice genome. This study will contribute to the improvement of the radiation mutation breeding research in genetic and genomic aspect.

Exposure to ionizing radiation is regarded as a kind of abiotic stresses that can change the expression of genes in living organisms. This study aimed on investigating the variations in gene expressions induced by two different types of irradiations with different doses, which were low linear energy transfer (LET) gamma rays (100, 200, and 400 Gy) and high LET ion-beams (20, 40, and 80 Gy) on rice. RNA sequencing was carried out using the Illumina HiSeq-2500 platform. The average amount of reads were 4.8 Gb per individual, and 5 to 8% of the reads were removed after quality control. More than 90% of the RNA-seq reads were mapped to the rice reference genome sequence (IRGSP-1.0). A total of 247 differentially expressed genes (DEGs) were identified by comparison of the gene expression levels between the wildtype and the irradiated individuals. The 247 DEGs were divided into five modules and 27 intra-modular hub genes were found using the weighted correlation network analysis (WGCNA) method. The MEturquiose module had the most number of genes with 75 related to carbohydrate and small molecule metabolic processes. The co-expression network reconstructed using ARACNE (algorithm for reconstruction of accurate cellular networks) showed specific up- or down-regulation of the genes in each module according to the types and doses of radiation. This study will contribute to understanding the gene expression responses to ionizing irradiation.

Perilla frutescens (L.) is an annual herbaceous and ornamental plant in the Lamiaceae family. Perilla frutescens (L.) Britt.cv.Chookyoupjaso were irradiated using a 200 Gy gamma ray in 1995. By HPLC analysis, this new cultivar significantly induced isoegomaketone content compared with ‘Chookyoupjaso’ control. The phenotypical difference was the changed leaf color of the ‘Atom-Ketone’ from violet to green. The yield potential of this cultivar (106 kg/10a) was 1.83 folds higher than that of ‘Chookyoupjaso’ (57.65 kg/10a). The methanol extracts of ‘Atom-Ketone’ inhibit nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells. This extract was further partitioned using ethyl acetate (EtOAc), butanol (BuOH), and water. The EtOAc fraction (EF-Atom-Ketone) was evaluated for antiinflammatory activities. These results indicated that the EF-Atom-Ketone reduced NO production by inhibiting inducible nitric oxide synthase (iNOS) expression. The EF-Atom-Ketone treatment also significantly diminished expression of MCP-1 and IL-6. Therefore, ‘Atom-Ketone’ reveals the potential therapeutic use of bioactive

Perilla frutescens (L.) is an edible plant, not only used as s food ingredient, but also in skin cream, soaps, and medicinal preparetions. ‘Atom-Ros’, a perilla (Perilla frutescenc (L.) Britt. cv. Chookyoupjaso was developed in 1995 by 200 Gy gamma irradiation-mutagenesis. This new cultivar has high rosmarinic acid content more than two fold compare with ‘Chookyoupjaso’ control. The observed phenotypical difference was changed leaf color of the ‘Atom-Ketone’ from violet to green. The yield potential of this cultivar (123.5 kg/10a) was 2.14 fold higher than that of ‘Chookyoupjaso’ (57.65 kg/10a). The methanol extracts of ‘Atom-Ros’ were tested for inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophase cells. Atom-Ros showed significant inhibition of NO production. This rosmarinic acid extracted from ‘Atom-Ros’ has a good potential to be developed as an antioxidant agent.

Copy number variations (CNVs) are considered major sources of genetic variation, and CNVs may influence phenotypic variation and gene expression. To detect CNVs, rice seeds were exposed with 100～400 Gy of gamma-ray (GA, 60Co), cosmic-ray (CR) by Russia ISS, and 30 and 40 Gy of ion beam (IB, 220 Mev carbon ion). After the exposed rice seeds were cultured in 1/2 MS medium for 14 days, they were used for array-based Comparative genomic hybridization (CGH) analysis using Agilent’s RICE CGH array. As a results, the highest number of CNVs (Gain 808 and Loss 24,080) were detected in the CR treatment, whereas GA100 (100 Gy of GA) was identified the least CNVs. Compared individual chromosome, the chromosome 8 and 11 were identified the highest CNVs, the chromosome 3 had the least CNVs. Most of identified CNVs existed in the range of 10～500kb. In particular, the same CNV locations among different types of ionizing radiation were observed in chromosome 12, and these CNVs contained the commonly 5 amplified genes, containing retrotransposon protein, NADH-ubiquinone oxidoreductase chain 3, heavy metal transport/detoxification protein domain containing protein, and 2 unknown proteins. Other studies were reported that Ty1 (Long Terminal Repeat-retrotransposon family 1) transcription and retrotransposition were induced by different environmental stresses such as ionizing radiation, UV-light exposure, DNA damage and nutrient starvation in Saccharomyces cerevisiae. Our results also show that retrotransposon protein (LOC_Os12g34016) was specifically amplified by different types of ionizing radiation.

VitE (tocotrienols and tocopherols) are micronutrients with antioxidant properties synthesized by photosynthetic bacteria and plants that play important roles in animal and human nutrition. A new mutant line, T1001-1, was isolated from in vitro mutagenized population by ionizing radiation and shown to have increased VitE contents. The total VitE content was 26% increased in the T1001-1 mutant seeds compare with cv. Dongan (wild-type). In addition, we showed that the mutant confers retarded seedling growth during the early seedling growth stage in rice. To study the molecular mechanism of VitE biosynthesis, we used the rice microarray to identify genes that are upor down-regulated in T1001-1 mutant. In addition, we identified differentially regulated pathway using MapMan analysis, which provides deep insight into changes in transcript and metabolites. Our results enhanced the transcription of genes involved in starch and lipid metabolism in T1001-1 mutant. To identify the molecular mechanisms of the events involving transcription factors in tocopherol accumulation, we compared the expression patterns of transcription factors. The AP2-EREBP, WRKY, C2H2 transcription factor were up-regulated, whereas the MYB family was down-regulated in T1001-1 mutant. Our results demonstrate change of important transcript in high level of VitE accumulating rice mutant.

Ionizing radiation is known to cause chromosomal alterations such as inversions and deletions and affects gene expression within the plant genome. To monitor the genome-wide transcriptome changes by ionizing radiation, we used rice Affimetrix GeneChip microarray to identify genes that are up- or down regulated by gamma-ray (200 Gy, 60Co source), cosmic-ray and ion beam (40 Gy, 220 MeV carbon ion). The overall expression patterns between gamma-ray and ion beam were similar but cosmic-ray was regulated differently. Combined results from all 3 radiations identified 27 up-regulated genes and 188 down regulated genes. These results mean the induction of similar mechanism changes in treatments of gamma ray and ion beam. However the different expression in treatment of cosmic-ray might be due to the other environmental conditions. Among the commonly up- or down- regulated genes, we chose highly up- or down- regulated several genes and confirmed its regulation in response to ionizing radiation exposure by RT-PCR analysis. Moreover, we showed that specific co-expression networks of candidate radio marker genes by ARACNE algorithm. Our results present profiles of gene expression related to different ionizing radiation and marker gene to predict sensitivity to ionizing radiation, such as GS (glutelin subunit) and FBX322.