Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd·kg −1 in the grain compared with a mean of 1.73 mg Cd·kg −1 in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ionbeam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

Recently, heavy ions or ion beams have been used to generate new mutants or varieties, especially in higher plants. It has been found that ion beams show high relative biological effectiveness (RBE) of growth inhibition, lethality, and so on, but the characteristics of ion beams on mutation have not been clearly elucidated. To understand the effect of ion beams on mutation induction, mutation rates were investigated using visible known Arabidopsis mutant phenotypes, indicating that mutation frequencies induced by carbon ions were 20-fold higher than by electrons. In chrysanthemum and carnation, flower-color and flower-form mutants, which are hardly produced by gamma rays or X rays, were induced by ion beams. Novel mutants and their responsible genes, such as UV-B resistant, serrated petals and sepals, anthocyaninless, etc. were induced by ion beams. These results indicated that the characteristics of ion beams for mutation induction are high mutation frequency and broad mutation spectrum and therefore, efficient induction of novel mutants. On the other hand, PCR and sequencing analyses showed that half of all mutants induced by ion beams possessed large DNA alterations, while the rest had point-like mutations. Both mutations induced by ion beams had a common feature that deletion of several bases were predominantly induced. It is plausible that ion beams induce a limited amount of large and irreparable DNA damage, resulting in production of a null mutation that shows a new mutant phenotype.

Legume plants tightly control the development and number of symbiotic root nodules. In Lotus japonicus, this regulation requires HAR1 (a CLAVATA1-like receptor kinase) in the shoots, suggesting that a long-distance communication between the shoots and the roots may exist. To better understand its molecular basis, we isolated and characterized a novel hypernodulating mutant of L. japonicus named too much love (tml). Compared with the wild type, tml mutants produced much more nodules which densely covered a wider range of the roots. Reciprocal grafting showed that tml hypernodulation is determined by the root genotype. Moreover, grafting a har1 shoot onto a tml rootstock did not exhibit any obvious additive effects on the nodule number, which was further supported by double mutational analysis. These observations indicate that a shoot factor HAR1 and a root factor TML participate in the same genetic pathway which governs the long-distance signaling of nodule number control. We also showed that the inhibitory effect of TML on nodulation is likely to be local. Therefore, TML may function downstream of HAR1 and the gene product TML might serve as a receptor or mediator of unknown mobile signal molecules that are transported from the shoots to the roots.

To investigate UV light response mechanisms in higher plants, we isolated a UV light-sensitive mutant, rev3-1 , in Arabidopsis. The root growth of rev3-1 was inhibited after UV-B irradiation under both light and dark conditions. We found that chromosome 1 of rev3-1 was broken at a minimum of three points, causing chromosome inversion and translocation. A gene disrupted by this rearrangement encoded the catalytic subunit of DNA polymerase ( AtREV3 ), which is thought to be involved in translesion synthesis. The rev3-1 seedlings also were sensitive to ␥ -rays and mitomycin C, which are known to inhibit DNA replication. Incorporation of bromodeoxyuridine after UV-B irradiation was less in rev3-1 than in the wild type. These results indicate that UV light-damaged DNA interrupted DNA replication in the rev3-1 mutant, leading to the inhibition of cell division and root elongation.

Gamma rays are the most frequently used ionizing radiation in plant mutagenesis; however, few studies are available on the characteristics of mutations at a genome-wide level. Here, we quantitatively and qualitatively characterized the mutations induced by acute/chronic gamma ray irradiation in Arabidopsis. The data were then compared with those previously obtained for carbon ion irradiation. In the acute irradiation of dry seeds at the same effective survival dose, gamma rays and carbon ions differed substantially, with the former inducing a significantly greater number of total mutation events, while the number of gene-affecting mutation events did not differ between the treatments. This may result from the gamma rays predominantly inducing single-base substitutions, while carbon ions frequently induced deletions ≥2 bp. Mutation accumulation lines prepared by chronic gamma irradiation with 100-500 mGy/h in five successive generations showed higher mutation frequencies per dose compared with acute irradiation of dry seeds. Chronic gamma ray irradiation may induce larger genetic changes compared with acute gamma ray irradiation. In addition, the transition/transversion ratio decreased as the dose rate increased, suggesting that plants responded to very low dose rates of gamma rays (∼1 mGy/h), even though the overall mutation frequency did not increase. These data will aid our understanding of the effects of radiation types and be useful in selecting suitable radiation treatments for mutagenesis.

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SummaryWe have isolated and characterized a new ultraviolet B (UV-B)-resistant mutant, uvi4 (UV-B-insensitive 4), of Arabidopsis. The fresh weight (FW) of uvi4 plants grown under supplemental UV-B light was more than twice that of the wild-type. No significant difference was found in their ability to repair the UV-B-induced cyclobutane pyrimidine dimers, or in the amount of UV-B absorptive compounds, both of which are wellknown factors that contribute to UV sensitivity. Positional cloning revealed that the UVI4 gene encodes a novel basic protein of unknown function. We found that the hypocotyl cells in uvi4 undergo one extra round of endoreduplication. The uvi4 mutation also promoted the progression of endo-reduplication during leaf development. The UVI4 gene is expressed mainly in actively dividing cells. In the leaves of P UVI4 ::GUS plants, the GUS signal disappeared in basipetal fashion as the leaf developed. The total leaf blade area was not different between uvi4 and the wild-type through leaf development, while the average cell area in the adaxial epidermis was considerably larger in uvi4, suggesting that the uvi4 leaves have fewer but larger epidermal cells. These results suggest that UVI4 is necessary for the maintenance of the mitotic state, and the loss of UVI4 function stimulated endo-reduplication. Tetraploid Arabidopsis was hyper-resistant to UV-B compared to diploid Arabidopsis, suggesting that the enhanced polyploidization is responsible for the increased UV-B tolerance of the uvi4 mutant.

Ionizing radiation including heavy-ion beams has been widely used in mutation breeding. Dry seeds, seedlings, and cultured tissues are often used for mutagenesis; however, little is known about the differences in induced mutations among them. Here, we examined the characteristics of mutations using randomly chosen Arabidopsis M2 plants derived from dry seeds and seedlings irradiated with carbon ions. The mutation frequency was 1.4–1.9 times higher in dry-seed irradiation than in seedling irradiation. This difference was mainly due to the three-times higher frequency of insertions and deletions (InDels) in dry-seed irradiation than in seedling irradiation. This difference increased the proportion of mutations predicted to affect gene function among all mutations identified by whole genome re-sequencing. Our results demonstrate that the physiological status of plant tissue greatly affects the characteristics of mutations induced by ionizing radiation, and that dry seeds are more suitable materials than seedlings for inducing loss-of-function mutations. The results also showed that single base deletions often occurred in homopolymeric sequences, while InDels larger than 2–3 bp often occurred in or near polynucleotide-repeat or microhomologous sequences. Interestingly, microhomology was less commonly found around large deletions (≥50 bp), suggesting that the rejoining process differs depending on the deletion size.