Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana.

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via assembly of the FtsZ ring.

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.

Distinctive development of embryo and endosperm caused by male gametes irradiated with carbon-ion beam.

KEY MESSAGE: In Cyrtanthus mackenii, development of embryo and endosperm were differentially affected by fertilization of male gametes with DNA damage and mutations. Pollen irradiation with ionizing radiations has been applied in plant breeding and genetic research, and haploid plant induction has mainly been performed by male inactivation with high-dose irradiation. However, the fertilization process of irradiated male gametes and the early development of embryo and endosperm have not received much attention. Heavy-ion beams, a type of radiation, have been widely applied as effective mutagens for plants and show a high mutation rate even at low-dose irradiation. In this study, we analyzed the effects of male gametes of Cyrtanthus mackenii irradiated with a carbon-ion beam at low doses on fertilization. In immature seeds derived from the pollination of irradiated pollen grains, two types of embryo sacs were observed: embryo sac with a normally developed embryo and endosperm and embryo sac with an egg cell or an undivided zygote and an endosperm. Abnormalities in chromosome segregation, such as chromosomal bridges, were observed only in the endosperm nuclei, irrespective of the presence or absence of embryogenesis. Therefore, in Cyrtanthus, embryogenesis is strongly affected by DNA damage or mutations in male gametes. Moreover, various DNA contents were detected in the embryo and endosperm nuclei, and endoreduplication may have occurred in the endosperm nuclei. As carbon-ion irradiation causes chromosomal rearrangements even at low doses, pollen irradiation can be an interesting tool for studying double fertilization and mutation heritability.

Characterization of a novel mutant with inhibition of storage root formation in sweet potato.

Sweet potato is a widely cultivated crop with storage roots. Although many studies have been conducted on the mechanism of its storage root formation, the details have not been fully elucidated. We screened mutant lines with inhibition of storage root formation to clarify parts of the mechanism. In this study, the process of storage root formation in one of the mutant lines, C20-8-1, was investigated. The inhibition of storage root formation was observed during the early stages of growth. The roots in C20-8-1 did not show histological differences compared to those in wild type. The transition from fibrous roots to pencil roots, which are the developmental stages prior to mature storage root formation, was delayed or inhibited in C20-8-1. The upregulation of starch biosynthesis-related genes and downregulation of lignin biosynthesis genes with storage root swelling were not confirmed in the root of C20-8-1 during the developmental transition stage, suggesting that most of the roots in C20-8-1 are in the pre-transition state toward the storage root swelling. C20-8-1 showed a mutant phenotype during the critical period of storage root swelling initiation, and further clarification of this mutation is expected to provide new insights into storage root formation.

Effect of Growth Stages on Anthocyanins and Polyphenols in the Root System of Sweet Potato.

The storage roots of purple-fleshed sweet potato contain a variety of anthocyanins and polyphenols. Little is known about changes in the total content and composition of anthocyanins and polyphenols in the early growth stages of the root system. In this study, we investigated the changes in anthocyanins and polyphenols in the root system of purple-fleshed sweet potato cultivars at 15, 30, 45, and 60 days after transplant (DAT). Unexpectedly, the highest percentage of acylated anthocyanins in three purple-fleshed cultivars among all growth stages was at 15 DAT. On the other hand, the total polyphenol content in the early growth stages of the root system increased rapidly toward 45 DAT, just before the beginning of storage root enlargement, and then decreased rapidly as the storage roots began to enlarge. These data indicate that the early growth stage of the root system is a critical time. This timing may present a strategy to maximize the accumulation of polyphenols with high antioxidant activity, as well as acylated anthocyanins, to protect against abiotic and biotic stresses.

Comparison of Proanthocyanidin Content in Rabbiteye Blueberry (Vaccinium virgatum Aiton) Leaves and the Promotion of Apoptosis against HL-60 Promyelocytic Leukemia Cells Using 'Kunisato 35 Gou' Leaf Extract.

Polyphenol-rich rabbiteye blueberry (Vaccinium virgatum Aiton) leaves have attracted attention as a food material. In this study, we compared the total polyphenols, total proanthocyanidin content, and antioxidant activity of the leaves of 18 blueberry varieties and investigated the seasonal variation in polyphenols. We also evaluated the anti-cancer cell proliferation properties of the rabbiteye blueberry leaf specific cultivar 'Kunisato 35 Gou'. Rabbiteye blueberry leaves had significantly higher total polyphenol and total proanthocyanidin values than northern highbush blueberry and southern highbush blueberry leaves. The antioxidant activity of blueberry leaves was highly positively correlated with both the total polyphenol and total proanthocyanidin content. Variations were observed in the total polyphenol and total proanthocyanidin content of rabbiteye blueberry leaves harvested at different points in the growing season; leaves collected in fall to winter contained more epicatechin in addition to proanthocyanidins. In the evaluation of anti-cancer cell proliferation properties against HL-60 promyelocytic leukemia cells, the September-harvested extracts of rabbiteye blueberry 'Kunisato 35 Gou' showed strong properties, and the use of an FITC Annexin V apoptosis detection kit with propidium iodide confirmed that this HL-60 cell death occurred via apoptosis. Limiting the harvest time would make rabbiteye blueberry leaves a more functional food ingredient.

Comprehensive effects of heavy-ion beam irradiation on sweet potato (Ipomoea batatas [L.] Lam.).

Sweet potato is a major root crop with nutritious tuberous roots. The mechanism of tuberous root development has not yet been adequately elucidated. Genetic resources are required to develop the molecular understanding of sweet potato. Heavy-ion beams were applied to hexaploid sweet potato for an increase in genetic variation, after which the comprehensive effects of heavy-ion beam irradiation were investigated. In vitro cultured shoots with an axillary bud of 'Beniharuka' were irradiated with Ar-ions at a dose of 1-5 Gy and C-ions at a dose of 5-20 Gy, and three irradiated lines were separated from each irradiated shoot. The shoot regeneration was inhibited at high doses of each ion irradiation. Ar-ion irradiation had an especially high biological effect on shoot regeneration. A total of 335 lines were obtained, consisting of 104 and 231 lines derived from Ar- and C-ion irradiation, respectively. The change in the DNA content of the lines was analyzed by flow cytometry to evaluate the irradiation-induced damage to the DNA. The two lines demonstrated significant differences in the DNA content and changes at the chromosome level. The screening for the morphological mutants was conducted in the field. Some irradiated lines showed inhibited or no tuberous root phenotype as mutant candidates. Additionally, the high-yield mutant candidates were dominated by Ar-ion irradiation. It was indicated that heavy-ion beam mutagenesis is effective in broadening the range of the phenotypes corresponding to tuberous root formation in hexaploid sweet potato.

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.

LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa.

PURPOSE: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora. MATERIALS AND METHODS: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined. RESULTS: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET). CONCLUSIONS: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

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.

AMAP: A pipeline for whole-genome mutation detection in Arabidopsis thaliana.

Detection of mutations at the whole-genome level is now possible by the use of high-throughput sequencing. However, determining mutations is a time-consuming process due to the number of false positives provided by mutation-detecting programs. AMAP (automated mutation analysis pipeline) was developed to overcome this issue. AMAP integrates a set of well-validated programs for mapping (BWA), removal of potential PCR duplicates (Picard), realignment (GATK) and detection of mutations (SAMtools, GATK, Pindel, BreakDancer and CNVnator). Thus, all types of mutations such as base substitution, deletion, insertion, translocation and chromosomal rearrangement can be detected by AMAP. In addition, AMAP automatically distinguishes false positives by comparing lists of candidate mutations in sequenced mutants. We tested AMAP by inputting already analyzed read data derived from three individual Arabidopsis thaliana mutants and confirmed that all true mutations were included in the list of candidate mutations. The result showed that the number of false positives was reduced to 12% of that obtained in a previous analysis that lacked a process of reducing false positives. Thus, AMAP will accelerate not only the analysis of mutation induction by individual mutagens but also the process of forward genetics.

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.

Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement.

Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves ß-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H(+)-PPase activity is required for gluconeogenesis. Lack of H(+)-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi) levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE). Here, we mutagenized fugu5-1 seeds with (12)C(6+) heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s) involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm), similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2) as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1, and A#3-1; fugu5-1. Taken together, these results suggest that defects in either H(+)-PPase or ECH2 compromise cell proliferation due to defects in mobilizing seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post-mitotic cell expansion stage of cotyledon development, probably by converting indolebutyric acid to indole acetic acid.

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.

The conflict between cell proliferation and expansion primarily affects stem organogenesis in Arabidopsis.

Plant shoot organs such as stems, leaves and flowers are derived from specialized groups of stem cells organized at the shoot apical meristem (SAM). Organogenesis involves two major processes, namely cell proliferation and differentiation, whereby the former contributes to increasing the cell number and the latter involves substantial increases in cell volume through cell expansion. Co-ordination between the above processes in time and space is essential for proper organogenesis. To identify regulatory factors involved in proper organogenesis, heavy-ion beam-irradiated de-etiolated (det) 3-1 seeds have been used to identify striking phenotypes in the A#26-2; det3-1 mutant. In addition to the stunted plant stature mimicking det3-1, the A#26-2; det3-1 mutant exhibited stem thickening, increased floral organ number and a fruit shape reminiscent of clavata (clv) mutants. DNA sequencing analysis demonstrated that A#26-2; det3-1 harbors a mutation in the CLV3 gene. Importantly, A#26-2; det3-1 displayed cracks that randomly occurred on the main stem with a frequency of approximately 50%. Furthermore, the double mutants clv3-8 det3-1, clv1-4 det3-1 and clv2-1 det3-1 consistently showed stem cracks with frequencies of approximately 97, 38 and 35%, respectively. Cross-sections of stems further revealed an increase in vascular bundle number, cell number and size in the pith of clv3-8 det3-1 compared with det3-1. These findings suggest that the stem inner volume increase due to clv mutations exerts an outward mechanical stress; that in a det3-1 background (defective in cell expansion) resulted in cracking of the outermost layer of epidermal cells.

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.

DNA damage response in male gametes of Cyrtanthus mackenii during pollen tube growth.

Male gametophytes of plants are exposed to environmental stress and mutagenic agents during the double fertilization process and therefore need to repair the DNA damage in order to transmit the genomic information to the next generation. However, the DNA damage response in male gametes is still unclear. In the present study, we analysed the response to DNA damage in the generative cells of Cyrtanthus mackenii during pollen tube growth. A carbon ion beam, which can induce DNA double-strand breaks (DSBs), was used to irradiate the bicellular pollen, and then the irradiated pollen grains were cultured in a liquid culture medium. The male gametes were isolated from the cultured pollen tubes and used for immunofluorescence analysis. Although inhibitory effects on pollen tube growth were not observed after irradiation, sperm cell formation decreased significantly after high-dose irradiation. After high-dose irradiation, the cell cycle progression of generative cells was arrested at metaphase in pollen mitosis II, and phosphorylated H2AX (γH2AX) foci, an indicator of DSBs, were detected in the majority of the arrested cells. However, these foci were not detected in cells that were past metaphase. Cell cycle progression in irradiated generative cells is regulated by the spindle assembly checkpoint, and modification of the histones surrounding the DSBs was confirmed. These results indicate that during pollen tube growth generative cells can recognize and manage genomic lesions using DNA damage response pathways. In addition, the number of generative cells with γH2AX foci decreased with culture prolongation, suggesting that the DSBs in the generative cells are repaired.

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.

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana.

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1-green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backwards. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1-GFP-labeled plastids, thus validating the use of FtsZ1-GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.

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.