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Proton ions are expected to be used as a discriminative radiation source to induce different kinds of mutations than those produced by γ-rays and carbon ions; however, there is little systemic information about radiosensitivity in plants. We analyzed the LD30, LD50, and RD50 values in response to proton ions and γ-rays using 20 plant species. Plant seeds were irradiated, and growth responses were measured one month after planting, except for cymbidium, for which in vitro rhizomes were irradiated. The rhizomes were analyzed at six and nine months after subculturing. Resistance to proton ions and γ-rays was observed in Chinese cabbage, watermelon, and melon, while Japanese atractylodes, naked barley, and lentil were susceptible. Plants belonging to the Brassicaceae and Cucurbitaceae families were highly resistant to radiation, and plants belonging to the Compositae and Poaceae families were highly susceptible. In addition, plants with genome sizes greater than 8,000 Mbp were highly sensitive to radiation, but there was no clear relationship between radiosensitivity and genome size in plants with genomes smaller than 2,500 Mbp. The biological effectiveness of proton ions was greater than that of γ-rays in 16 plant species, indicating that they could be used as a discriminative radiation source to induce mutations compared with γ-rays. [ABSTRACT FROM AUTHOR]

Protons may have contributed to the evolution of plants as a major component of cosmic-rays and also have been used for mutagenesis in plants. Although the mutagenic effect of protons has been well-characterized in animals, no comprehensive phenotypic and genomic analyses has been reported in plants. Here, we investigated the phenotypes and whole genome sequences of Arabidopsis M2 lines derived by irradiation with proton beams and gamma-rays, to determine unique characteristics of proton beams in mutagenesis. We found that mutation frequency was dependent on the irradiation doses of both proton beams and gamma-rays. On the basis of the relationship between survival and mutation rates, we hypothesized that there may be a mutation rate threshold for survived individuals after irradiation. There were no significant differences between the total mutation rates in groups derived using proton beam or gamma-ray irradiation at doses that had similar impacts on survival rate. However, proton beam irradiation resulted in a broader mutant phenotype spectrum than gamma-ray irradiation, and proton beams generated more DNA structural variations (SVs) than gamma-rays. The most frequent SV was inversion. Most of the inversion junctions contained sequences with microhomology and were associated with the deletion of only a few nucleotides, which implies that preferential use of microhomology in non-homologous end joining was likely to be responsible for the SVs. These results show that protons, as particles with low linear energy transfer (LET), have unique characteristics in mutagenesis that partially overlap with those of low-LET gamma-rays and high-LET heavy ions in different respects. [ABSTRACT FROM AUTHOR]

A total of 2561 gamma ray-induced mutants (M7) were evaluated in six abiotic stress treatments, and then principal component analysis was performed to score their phenotypes. Resequencing was performed on 123 selected rice mutants and wild-type rice to assess the structural changes in the genomes of these mutants, such as single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels). A total of 5160 (avg. 87.3 per plant) SNPs and 7195 (avg. 121.9) InDels were observed in the 200-Gy lines, whereas 5650 (avg. 88.3) SNPs and 7676 (avg. 119.9) InDels were found in the 300-Gy lines. We found that one candidate gene (Os02g0528900, which is similar to the gene for pleiotropic drug resistance (PDR)-like ATP-binding cassette (ABC) transporter) contained two SNPs that were included in principal component 4 via a genome-wide association study. The 300-42 line had the lowest phenotype score against heat, possibly suggesting the existence of a relationship between these SNPs and heat stress. Co-expression analysis of the candidate gene involved in responses to toxic substances and the chemical stimulation of significant biological processes. Therefore, the studied targeting induced local lesions in genome (TILLING) population could be a useful source of mutations in the breeding of new genetic resources for forward and reverse genetics. [ABSTRACT FROM AUTHOR]

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In this study, we performed whole genome resequencing by Illumina HiSeq to identify changes in flowering genes using an early-flowering phenotype of soybean mutant line Josaengserori (JS) derived from Korean landrace, Seoritae (SR), and we obtained mapped reads of 131,769,690 and 167,669,640 bp in JS and SR, respectively. From the whole genome sequencing results between JS and SR, we identified 332,821 polymorphic SNPs and 65,178 indels, respectively. Among these, 30 flowering genes were in SNPs and 25 were in indels. Among 30 flowering genes detected in SNPs, Glyma02g33040, Glyma06g22650, Glyma10g36600, Glyma13g01290, Glyma14g10530, Glyma16g01980, Glyma17g11040, Glyma18g53690, and Glyma20g29300 were non-synonymous substitutions between JS and SR. Changes in Glyma10g36600 ( GI), Glya02g33040 ( AGL18), Glyma17g11040 ( TOC1), and Glyma14g10530 ( ELF3) in JS affected the expression of GmFT2a and resulted in early flowering. These results provide insight into the regulatory pathways of flowering in soybean mutants and help to improve our knowledge of soybean mutation breeding. [ABSTRACT FROM AUTHOR]

With 5 figures and 1 table Abstract One thousand three hundred and fifty homologous rice M8 mutant lines resistant to 5-methyltryptophan, a tryptophan analogue, were selected from embryogenic calli irradiated with 30-120 Gy of gamma rays. The mutant rice would be compromised in tryptophan homoeostasis in the cell if a negative feedback by anthranilate synthase (AS) in tryptophan biosynthesis was altered by gamma irradiation, consequently causing tryptophan over-accumulation. Therefore, the correlation between traits of over-accumulated tryptophan and the alteration of AS by mutagenesis including single-nucleotide polymorphisms (SNPs) would enable the visualization and selection of over-accumulated mutant lines from the rice mutant population. Targeting Induced Local Lesions IN Genome analysis with five fluorescent-labelled primer sets covering the OASA1 locus resulted in the selection of three rice mutant lines, MRVII-9, MRVII-33 and MRVII-37. The tryptophan contents in the seed storage protein of the three mutant lines were elevated when compared with the wild-type cv. 'Dongan'. Genomic DNA of OASA1 was re-sequenced and validated at the SNP and other polymorphism sites. Three mutant lines (MRVII-9, MRVII-33 and MRVII-37) not only contained more than five SNP sites in the total regions of OASA1 gene, but also contained SNPs in coding regions that resulted in amino acid changes such as E241G, A560G and V495F/S496F, respectively. The SNPs, which resulted in amino acid changes, may affect the enhanced tryptophan content in the seed storage protein. These different features could utilize the selection of mutant lines with over-accumulated tryptophan from large rice mutant populations. [ABSTRACT FROM AUTHOR]

Anthocyanins (a subclass of flavonoids) and flavonoids are crucial determinants of flower color and substances of pharmacological efficacy, respectively, in chrysanthemum. However, metabolic and transcriptomic profiling regarding flavonoid accumulation has not been performed simultaneously, thus the understanding of mechanisms gained has been limited. We performed HPLC-DAD-ESI-MS (high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry) and transcriptome analyses using "ARTI-Dark Chocolate" (AD), which is a chrysanthemum mutant cultivar producing dark-purple ray florets, and the parental cultivar "Noble Wine" for metabolic characterization and elucidation of the genetic mechanism determining flavonoid content. Among 26 phenolic compounds identified, three cyanidins and eight other flavonoids were detected only in AD. The total amounts of diverse flavonoids were 8.0 to 10.3 times higher in AD. Transcriptome analysis showed that genes in the flavonoid biosynthetic pathway were not up-regulated in AD at the early flower stage, implying that the transcriptional regulation of the pathway did not cause flavonoid accumulation. However, genes encoding post-translational regulation-related proteins, especially F-box genes in the mutated gene, were enriched among down-regulated genes in AD. From the combination of metabolic and transcriptomic data, we suggest that the suppression of post-translational regulation is a possible mechanism for flavonoid accumulation in AD. These results will contribute to research on the regulation and manipulation of flavonoid biosynthesis in chrysanthemum. [ABSTRACT FROM AUTHOR]

Ionizing radiation combined with in vitro tissue culture has been used for development of new cultivars in diverse crops. The effects of ionizing radiation on mutation induction have been analyzed on several orchid species, including Cymbidium. Limited information is available on the comparison of mutation frequency and spectrum based on phenotypes in Cymbidium species. In addition, the stability of induced chimera mutants in Cymbidium is unknown. In this study, we analyzed the radiation sensitivity, mutation frequency, and spectrum of mutants induced by diverse γ-ray treatments, and analyzed the stability of induced chimera mutants in the Cymbidium hybrid cultivars RB003 and RB012. The optimal γ-irradiation conditions of each cultivar differed as follows: RB003, mutation frequency of 4.06% (under 35 Gy/4 h); RB012, 1.51% (20 Gy/1 h). Re-irradiation of γ-rays broadened the mutation spectrum observed in RB012. The stability of leaf-color chimera mutants was higher than that of leaf-shape chimeras, and stability was dependent on the chimera type and location of a mutation in the cell layers of the shoot apical meristem. These results indicated that short-term γ-irradiation was more effective to induce mutations in Cymbidium. Information on the stability of chimera mutants will be useful for mutation breeding of diverse ornamental plants. [ABSTRACT FROM AUTHOR]

Radiation randomly induces chromosomal mutations in plants. However, it was recently found that the frequency of flower-color mutants could be specifically increased by upregulating anthocyanin pathway gene expression before radiation treatments. The mechanisms of chlorophyll biosynthesis and degradation are active areas of plant study because chlorophyll metabolism is closely connected to photosynthesis. In this study, we determined the dark/light treatment conditions that resulted in upregulation of the expression levels of six chlorophyll pathway genes, uroporphyrinogen III synthase (HEMD), uroporphyrinogen III decarboxylase (HEME2), NADPH-protochlorophyllide oxidoreductase (POR) A (PORA), chlorophyll synthase (CHLG), chlorophyllase (CLH2), and red chlorophyll catabolite reductase (RCCR), and measured their effects on the γ-irradiation-induced frequencies of leaf-color mutants in two Cymbidium cultivars. To degrade chlorophyll in rhizomes, 60–75 days of dark treatment were required. To upregulate the expressions of chlorophyll pathway genes, 10 days of light treatment appeared to be optimal. Dark/light treatments followed by γ-irradiation increased chlorophyll-related leaf mutants by 1.4- to 2.0-fold compared with γ-ray treatment alone. Dark/light treatments combined with γ-irradiation increased the frequency of leaf-color mutants in Cymbidium, which supports the wider implementation of a plant breeding methodology that increases the mutation frequency of a target trait by controlling the expression of target trait-related genes. [ABSTRACT FROM AUTHOR]

In addition to attracting pollinators and protecting plants from photooxidative damages, carotenoids play important roles in signaling related to plant development and physiological changes through their cleaved derivatives, apocarotenoids. Yellow chrysanthemum flower petals [ Chrysanthemum morifolium (Ramat.)] are the result of a deleted or suppressed carotenoid cleavage dioxygenase gene ( CmCCD4a ). Therefore, investigating the global gene expression patterns in chrysanthemum mutants producing yellow petals represents an appropriate strategy to analyze the effects of signaling mediated by carotenoid derivatives. We compared the transcriptomes of chrysanthemum cultivars producing white petals with purple stripes (‘Noble Wine’) and its mutant producing yellow petals with purple stripes (‘ARTI-Yellow Star’). Read mapping and polymerase chain reaction analysis revealed that CmCCD4a is absent in the ARTI-Yellow Star genome, while at least three CmCCD4a gene family members are actively transcribed in ‘Noble Wine’ plants. These results indicate that the deletion of CmCCD4a may be responsible for the development of yellow petals in ARTI-Yellow Star flowers. During comparisons of gene expression patterns between Noble Wine and ARTI-Yellow Star, we detected numerous significantly up-regulated plastid protein-coding genes as well as significantly or mildly up-regulated carotenoid biosynthesis genes in ARTI-Yellow Star plants. The up-regulated plastid protein-coding genes belong to a broad range of functional categories related to translation, photosynthesis, reactive oxygen species scavenging, oxidative phosphorylation, transcription, processing, terpenoid metabolism, and chlorophyll metabolism. Our results present a possible relationship between CmCCD4a and regulation of plastid protein-coding genes. [ABSTRACT FROM AUTHOR]

Chrysanthemum is one of the most popular ornamental plants, whose petal colors are primarily determined by pigments including flavonoids/anthocyanins and carotenoids. To develop chrysanthemum cultivars with various petal colors, mutation breeding targeting alteration of pigmentation pattern has been performed. A radiation-induced mutant line, ‘ARTI-purple’, showed a flower color altered from the original bright pink to purple. In this study, we used suppression subtractive hybridization to analyze transcripts and characterize the differential gene expression of chrysanthemum petals between the mutant ‘ARTI-purple’ and its wild-type Chrysanthemum × morifolium cultivar ‘Argus’. One hundred and seventy-six genes were identified ( e- value ≤ 1e − 5) and classified based on sequence homology to genes with known or putative functions. The genes were categorized functionally by gene ontology analysis and their tentative pathways were confirmed using the TAIR database. The analyses revealed that these genes were related to carbohydrate metabolism, biosynthesis of secondary metabolites, and lipid metabolism. Six genes in a Kyoto encyclopedia of genes and genomes (KEGG) pathway which included the largest number of differentially expressed genes were selected for validation by quantitative PCR, and most of them showed higher expression levels compared with the wild-type. In addition, we isolated two novel clones (PC06E06 and PC08C09) having glutathione S -transferase (GST) family conserved domains and one clone (PC02G08) having a Multidrug and toxic compound extrusion (MATE) family conserved domain based on analysis using conserved domain database (CDD). The expressions of PC08C09 and PC02G08 were upregulated in ‘ARTI-purple’, which implies that anthocyanin accumulation pattern might be altered in mutant. In this study, we identified several differentially expressed genes between ‘Argus’ and ‘ARTI-purple’. The analysis suggested that several metabolic genes as well as glutathione S -transferases and MATEs might be involved in the control of flower pigmentation in chrysanthemum. [ABSTRACT FROM AUTHOR]

• Sucrose and plant hormones modulate the expression of genes in the anthocyanin pathway. • The expression of the genes was maximized at 18 h after treatment of 50 mM sucrose in chrysanthemum. • The expression of the genes was highly increased by treatment of sucrose with methyl jasmonate. • Pre-treatment of sucrose with methyl jasmonate increased the frequency of flower-color mutants induced by γ-irradiation. γ-rays have been used to develop many mutations in diverse ornamental plants, including chrysanthemum, of which most changes have been flower color. Flower color is determined by plant pigments, including anthocyanins, which are affected by mutation of genes in the anthocyanin biosynthesis pathway. Sucrose and plant hormones also crosstalk in complex signaling pathways and modulate anthocyanin biosynthesis. In this study, we determined the optimal sucrose and plant hormone treatments for expression of anthocyanin biosynthesis genes and compared the mutation frequency among sucrose, methyl jasmonate (MeJA), and/or γ-ray treatments in chrysanthemum. Anthocyanin biosynthesis genes were highly expressed with 50 mM sucrose treatment for 18 h. The highest expression levels of anthocyanin biosynthesis genes were observed under sucrose with MeJA treatment followed by MeJA treatment alone. Sucrose with abscisic acid (ABA) treatment, and ABA or GA 3 treatment alone, had no effect on expression of anthocyanin biosynthesis genes. Sucrose with MeJA pre-treatment followed by γ-irradiation resulted in the highest mutation frequency (14.5%) among all treatments and a 1.5-fold increase in mutation frequency compared with γ-ray treatment alone. The ratio of anthocyanin-related flower-color mutants with sucrose and/or MeJA pre-treatments followed by γ-irradiation was higher than that with γ-ray treatment alone. Sucrose and/or MeJA modulated the expression of anthocyanin biosynthesis genes and highly expressed genes may be affected more readily by γ-rays, thus resulting in an increased frequency of anthocyanin-related flower-color mutants in chrysanthemum. [ABSTRACT FROM AUTHOR]