Different mutational function of low- and high-linear energy transfer heavy-ion irradiation demonstrated by whole-genome resequencing of Arabidopsis mutants.

Heavy-ion irradiation is a powerful mutagen that possesses high linear energy transfer (LET). Several studies have indicated that the value of LET affects DNA lesion formation in several ways, including the efficiency and the density of double-stranded break induction along the particle path. We assumed that the mutation type can be altered by selecting an appropriate LET value. Here, we quantitatively demonstrate differences in the mutation type induced by irradiation with two representative ions, Ar ions (LET: 290 keV µm-1 ) and C ions (LET: 30.0 keV µm-1 ), by whole-genome resequencing of the Arabidopsis mutants produced by these irradiations. Ar ions caused chromosomal rearrangements or large deletions (≥100 bp) more frequently than C ions, with 10.2 and 2.3 per mutant genome under Ar- and C-ion irradiation, respectively. Conversely, C ions induced more single-base substitutions and small indels (<100 bp) than Ar ions, with 28.1 and 56.9 per mutant genome under Ar- and C-ion irradiation, respectively. Moreover, the rearrangements induced by Ar-ion irradiation were more complex than those induced by C-ion irradiation, and tended to accompany single base substitutions or small indels located close by. In conjunction with the detection of causative genes through high-throughput sequencing, selective irradiation by beams with different effects will be a powerful tool for forward genetics as well as studies on chromosomal rearrangements.

Comprehensive identification of mutations induced by heavy-ion beam irradiation in Arabidopsis thaliana.

Heavy-ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole-genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array-CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602,180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar-ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines.

Genomic view of heavy-ion-induced deletions associated with distribution of essential genes in Arabidopsis thaliana.

Heavy-ion beam, a type of ionizing radiation, has been applied to plant breeding as a powerful mutagen and is a promising tool to induce large deletions and chromosomal rearrangements. The effectiveness of heavy-ion irradiation can be explained by linear energy transfer (LET; keV µm-1). Heavy-ion beams with different LET values induce different types and sizes of mutations. It has been suggested that deletion size increases with increasing LET value, and complex chromosomal rearrangements are induced in higher LET radiations. In this study, we mapped heavy-ion beam-induced deletions detected in Arabidopsis mutants to its genome. We revealed that deletion sizes were similar between different LETs (100 to 290 keV µm-1), that their upper limit was affected by the distribution of essential genes, and that the detected chromosomal rearrangements avoid disrupting the essential genes. We also focused on tandemly arrayed genes (TAGs), where two or more homologous genes are adjacent to one another in the genome. Our results suggested that 100 keV µm-1 of LET is enough to disrupt TAGs and that the distribution of essential genes strongly affects the heritability of mutations overlapping them. Our results provide a genomic view of large deletion inductions in the Arabidopsis genome.

Characterization of a heavy-ion induced white flower mutant of allotetraploid Nicotiana tabacum.

KEY MESSAGE : We characterized a white flower mutant of allotetraploid N. tabacum as a DFR-deficient mutant; one copy of DFR has a cultivar-specific frameshift, while the other was deleted by heavy-ion irradiation. In most plants, white-flowered mutants have some kind of deficiency or defect in their anthocyanin biosynthetic pathway. Nicotiana tabacum normally has pink petals, in which cyanidin is the main colored anthocyanidin. When a relevant gene in the cyanidin biosynthetic pathway is mutated, the petals show a white color. Previously, we generated white-flowered mutants of N. tabacum by heavy-ion irradiation, which is accepted as an effective mutagen. In this study, we determined which gene was responsible for the white-flowered phenotype of one of these mutants, cv. Xanthi white flower 1 (xwf1). Southern blot analysis using a DNA fragment of the dihydroflavonol 4-reductase (DFR) gene as a probe showed that the xwf1 mutant lacked signals that were present in wild-type genomic DNAs. Sequence analysis demonstrated that one copy of the DFR gene (NtDFR2) was absent from the genome of the xwf1 mutant. The other copy of the DFR gene (NtDFR1) contained a single-base deletion resulting in a frameshift mutation, which is a spontaneous mutation in cv. Xanthi. Introduction of NtDFR2 cDNA into the petal limbs of xwf1 by particle bombardment resulted in production of the pink-colored cells, whereas introduction of NtDFR1 cDNA did not. These results indicate that xwf1 is a DFR-deficient mutant. One copy of NtDFR1 harbors a spontaneous frameshift mutation, while the other copy of NtDFR2 was deleted by heavy-ion beam irradiation.

Molecular nature of mutations induced by high-LET irradiation with argon and carbon ions in Arabidopsis thaliana.

Linear energy transfer (LET) is an important parameter to be considered in heavy-ion mutagenesis. However, in plants, no quantitative data are available on the molecular nature of the mutations induced with high-LET radiation above 101-124keVµm(-1). In this study, we irradiated dry seeds of Arabidopsis thaliana with Ar and C ions with an LET of 290keVµm(-1). We analyzed the DNA alterations caused by the higher-LET radiation. Mutants were identified from the M(2) pools. In total, 14 and 13 mutated genes, including bin2, egy1, gl1, gl2, hy1, hy3-5, ttg1, and var2, were identified in the plants derived from Ar- and C-ions irradiation, respectively. In the mutants from both irradiations, deletion was the most frequent type of mutation; 13 of the 14 mutated genes from the Ar ion-irradiated plants and 11 of the 13 mutated genes from the C ion-irradiated plants harbored deletions. Analysis of junction regions generated by the 2 types of irradiation suggested that alternative non-homologous end-joining was the predominant pathway of repair of break points. Among the deletions, the proportion of large deletions (>100bp) was about 54% for Ar-ion irradiation and about 64% for C-ion irradiation. Both current results and previously reported data revealed that the proportions of the large deletions induced by 290-keVµm(-1) radiations were higher than those of the large deletions induced by lower-LET radiations (6% for 22.5-30.0keVµm(-1) and 27% for 101-124keVµm(-1)). Therefore, the 290keVµm(-1) heavy-ion beams can effectively induce large deletions and will prove useful as novel mutagens for plant breeding and analysis of gene functions, particularly tandemly arrayed genes.

Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana.

BACKGROUND: Heavy-ion mutagenesis is recognised as a powerful technology to generate new mutants, especially in higher plants. Heavy-ion beams show high linear energy transfer (LET) and thus more effectively induce DNA double-strand breaks than other mutagenic techniques. Previously, we determined the most effective heavy-ion LET (LETmax: 30.0 keV µm(-1)) for Arabidopsis mutagenesis by analysing the effect of LET on mutation induction. However, the molecular structure of mutated DNA induced by heavy ions with LETmax remains unclear. Knowledge of the structure of mutated DNA will contribute to the effective exploitation of heavy-ion beam mutagenesis. RESULTS: Dry Arabidopsis thaliana seeds were irradiated with carbon (C) ions with LETmax at a dose of 400 Gy and with LET of 22.5 keV µm(-1) at doses of 250 Gy or 450 Gy. The effects on mutation frequency and alteration of DNA structure were compared. To characterise the structure of mutated DNA, we screened the well-characterised mutants elongated hypocotyls (hy) and glabrous (gl) and identified mutated DNA among the resulting mutants by high-resolution melting curve, PCR and sequencing analyses. The mutation frequency induced by C ions with LETmax was two-fold higher than that with 22.5 keV µm(-1) and similar to the mutation frequency previously induced by ethyl methane sulfonate. We identified the structure of 22 mutated DNAs. Over 80% of the mutations caused by C ions with both LETs were base substitutions or deletions/insertions of less than 100 bp. The other mutations involved large rearrangements. CONCLUSIONS: The C ions with LETmax showed high mutation efficiency and predominantly induced base substitutions or small deletions/insertions, most of which were null mutations. These small alterations can be determined by single-nucleotide polymorphism (SNP) detection systems. Therefore, C ions with LETmax might be useful as a highly efficient reverse genetic system in conjunction with SNP detection systems, and will be beneficial for forward genetics and plant breeding.

Double Mutant Analysis with the Large Flower Mutant, ohbana1, to Explore the Regulatory Network Controlling the Flower and Seed Sizes in Arabidopsis thaliana.

Two growth processes, cell proliferation and expansion, determine plant species-specific organ sizes. A large flower mutant in Arabidopsis thaliana, ohbana1 (ohb1), was isolated from a mutant library. In the ohb1 flowers, post-mitotic cell expansion and endoreduplication of nuclear DNA were promoted. The whole-genome resequencing and genetic analysis results showed that the loss of function in MEDIATOR16 (MED16), a mediator complex subunit, was responsible for the large flower phenotypes exhibited by ohb1. A phenotypic analysis of the mutant alleles in MED16 and the double mutants created by crossing ohb1 with representative large flower mutants revealed that MED16 and MED25 share part of the negative petal size regulatory pathways. Furthermore, the double mutant analyses suggested that there were genetically independent pathways leading to cell size restrictions in the floral organs which were not related to the MED complex. Several double mutants also formed larger and heavier seeds than the wild type and single mutant plants, which indicated that MED16 was involved in seed size regulation. This study has revealed part of the size-regulatory network in flowers and seeds through analysis of the ohb1 mutant, and that the size-regulation pathways are partially different between floral organs and seeds.

An Argon-Ion-Induced Pale Green Mutant of Arabidopsis Exhibiting Rapid Disassembly of Mesophyll Chloroplast Grana.

Argon-ion beam is an effective mutagen capable of inducing a variety of mutation types. In this study, an argon ion-induced pale green mutant of Arabidopsis thaliana was isolated and characterized. The mutant, designated Ar50-33-pg1, exhibited moderate defects of growth and greening and exhibited rapid chlorosis in photosynthetic tissues. Fluorescence microscopy confirmed that mesophyll chloroplasts underwent substantial shrinkage during the chlorotic process. Genetic and whole-genome resequencing analyses revealed that Ar50-33-pg1 contained a large 940 kb deletion in chromosome V that encompassed more than 100 annotated genes, including 41 protein-coding genes such as TYRAAt1/TyrA1, EGY1, and MBD12. One of the deleted genes, EGY1, for a thylakoid membrane-localized metalloprotease, was the major contributory gene responsible for the pale mutant phenotype. Both an egy1 mutant and F1 progeny of an Ar50-33-pg1 × egy1 cross-exhibited chlorotic phenotypes similar to those of Ar50-33-pg1. Furthermore, ultrastructural analysis of mesophyll cells revealed that Ar50-33-pg1 and egy1 initially developed wild type-like chloroplasts, but these were rapidly disassembled, resulting in thylakoid disorganization and fragmentation, as well as plastoglobule accumulation, as terminal phenotypes. Together, these data support the utility of heavy-ion mutagenesis for plant genetic analysis and highlight the importance of EGY1 in the structural maintenance of grana in mesophyll chloroplasts.

Arabidopsis EGY1 Is Critical for Chloroplast Development in Leaf Epidermal Guard Cells.

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two 40Ar17+ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells.

Linear Energy Transfer-Dependent Change in Rice Gene Expression Profile after Heavy-Ion Beam Irradiation.

A heavy-ion beam has been recognized as an effective mutagen for plant breeding and applied to the many kinds of crops including rice. In contrast with X-ray or γ-ray, the heavy-ion beam is characterized by a high linear energy transfer (LET). LET is an important factor affecting several aspects of the irradiation effect, e.g. cell survival and mutation frequency, making the heavy-ion beam an effective mutagen. To study the mechanisms behind LET-dependent effects, expression profiling was performed after heavy-ion beam irradiation of imbibed rice seeds. Array-based experiments at three time points (0.5, 1, 2 h after the irradiation) revealed that the number of up- or down-regulated genes was highest 2 h after irradiation. Array-based experiments with four different LETs at 2 h after irradiation identified LET-independent regulated genes that were up/down-regulated regardless of the value of LET; LET-dependent regulated genes, whose expression level increased with the rise of LET value, were also identified. Gene ontology (GO) analysis of LET-independent up-regulated genes showed that some GO terms were commonly enriched, both 2 hours and 3 weeks after irradiation. GO terms enriched in LET-dependent regulated genes implied that some factor regulates genes that have kinase activity or DNA-binding activity in cooperation with the ATM gene. Of the LET-dependent up-regulated genes, OsPARP3 and OsPCNA were identified, which are involved in DNA repair pathways. This indicates that the Ku-independent alternative non-homologous end-joining pathway may contribute to repairing complex DNA legions induced by high-LET irradiation. These findings may clarify various LET-dependent responses in rice.

Effect of high-LET Fe-ion beam irradiation on mutation induction in Arabidopsis thaliana.

Heavy-ion beams are powerful mutagens. They cause a broad spectrum of mutation phenotypes with high efficiency even at low irradiation doses and short irradiation times. These mutagenic effects are due to dense ionisation in a localised region along the ion particle path. Linear energy transfer (LET; keV·µm(-1)), which represents the degree of locally deposited energy, is an important parameter in heavy-ion mutagenesis. For high LET radiation above 290 keV∙µm(-1), however, neither the mutation frequency nor the molecular nature of the mutations has been fully characterised. In this study, we investigated the effect of Fe-ion beams with an LET of 640 keV∙µm(-1) on both the mutation frequency and the molecular nature of the mutations. Screening of well-characterised mutants (hy and gl) revealed that the mutation frequency was lower than any other ion species with low LET. We investigated the resulting mutations in the 4 identified mutants. Three mutants were examined by employing PCR-based methods, one of which had 2-bp deletion, another had 178 bp of tandemly duplication, and other one had complicated chromosomal rearrangements with variable deletions in size at breakpoints. We also detected large deletions in the other mutant by using array comparative genomic hybridisation. From the results of the analysis of the breakpoints and junctions of the detected deletions, it was revealed that the mutants harboured chromosomal rearrangements in their genomes. These results indicate that Fe-ion irradiation tends to cause complex mutations with low efficiency. We conclude that Fe-ion irradiation could be useful for inducing chromosomal rearrangements or large deletions.