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 ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

https://www.pnas.org/content/pnas/109/47/19166.full.pdf

Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice

Identification of the species origin of pollen has many applications, including assessment of plant–pollinator networks, reconstruction of ancient plant communities, product authentication, allergen monitoring, and forensics. Such applications, however, have previously been limited by microscopy-based identification of pollen, which is slow, has low taxonomic resolution, and has few expert practitioners. One alternative is pollen DNA barcoding, which could overcome these issues. Recent studies demonstrate that both chloroplast and nuclear barcoding markers can be amplified from pollen. These recent validations of pollen metabarcoding indicate that now is the time for researchers in various fields to consider applying these methods to their research programs. In this paper, we review the nascent field of pollen DNA barcoding and discuss potential new applications of this technology, highlighting existing limitations and future research developments that will improve its utility in a wide range of applications.

/pdf/pollen-dna-barcoding-current-applications-and-future-14s0udq8zv.pdf

Pollen DNA barcoding: current applications and future prospects.

There is increasing evidence that all cells sense mechanical forces in order to perform their functions. In animals, mechanotransduction has been studied during the establishment of cell polarity, fate, and division in single cells, and increasingly is studied in the context of a multicellular tissue. What about plant systems? Our goal in this review is to summarize what is known about the perception of mechanical cues in plants, and to provide a brief comparison with animals.

/pdf/life-behind-the-wall-sensing-mechanical-cues-in-plants-ysl6u6oqid.pdf

Life behind the wall: sensing mechanical cues in plants

The morphology of plant nuclei varies among different species, organs, tissues and cell types. However, mechanisms and factors involved in the maintenance of nuclear morphology are poorly understood. Because nuclei retain their shapes even after cytoskeletal inhibitor treatments both in vivo and in vitro, we assumed involvement of the nuclear lamina, which plays a critical role in the regulation of nuclear morphology in animals. The crude nuclear lamina fraction isolated from Arabidopsis thaliana leaves was analyzed by mass spectrometry, and putative nuclear lamina proteins were identified. Among their T-DNA insertion lines, nuclei of little nuclei1 (linc1) and linc4 disruptants were more spherical than those of wild-type plants. Because A. thaliana harbors four LINC genes, we prepared all single and linc1/4 and linc2/3 double disruptants. In leaf epidermal cells, the circularity index of the nucleus in all linc disruptants except linc3 was significantly higher than that in the wild-type plants. The extent of the effects of LINC1 and/or LINC4 disruption was significantly higher than that of the effects of LINC2 disruption. The nuclear area was significantly smaller in the linc1, linc4 and linc1/4 disruptants than in the wild-type plants. Regardless of the defects in nuclear morphology, all linc disruptants exhibited a normal ploidy level. In interphase cells, LINC1 and LINC4 were mainly localized to the nuclear periphery, whereas LINC2 was in the nucleoplasm and LINC3 was detected in both regions. From prometaphase to anaphase in mitotic root tip cells, LINC1 was co-localized with chromosomes, whereas other LINCs were dispersed in the cytoplasm.

LITTLE NUCLEI 1 and 4 Regulate Nuclear Morphology in Arabidopsis thaliana

The European Commission requested that the EFSA Panel on Genetically Modified Organisms deliver a scientific opinion related to risk assessment of plants developed using the zinc finger nuclease 3 technique (ZFN-3) which allows the integration of gene(s) in a predefined insertion site in the genome of the recipient species. Since other nucleases with a similar function to ZFN are considered in this opinion the term site-directed nuclease 3 (SDN-3) is used to describe the technique rather than ZFN-3 specifically. The EFSA GMO Panel considers that its guidance documents are applicable for the evaluation of food and feed products derived from plants developed using the SDN-3 technique and for performing an environmental risk assessment. However, on a case-by-case basis lesser amounts of event specific data may be needed for the risk assessment of plants developed using the SDN-3 technique. The EFSA GMO Panel compared the hazards associated with plants produced by the SDN-3 technique with those obtained by conventional plant breeding techniques and by currently used transgenesis. With respect to the genes introduced, the SDN-3 technique does not differ from transgenesis or from the other genetic modification techniques currently used, and can be used to introduce transgenes, intragenes or cisgenes. The main difference between the SDN-3 technique and transgenesis is that the insertion of DNA is targeted to a predefined region of the genome. Therefore, the SDN-3 technique can minimise hazards associated with the disruption of genes and/or regulatory elements in the recipient genome. Whilst the SDN-3 technique can induce off-target changes in the genome of the recipient plant these would be fewer than those occurring with most mutagenesis techniques. Furthermore, where such changes occur they would be of the same types as those produced by conventional breeding techniques.

https://www.zora.uzh.ch/id/eprint/75266/1/2943.pdf

Scientific opinion addressing the safety assessment of plants developed using Zinc Finger Nuclease 3 and other site-directed Nucleases with similar function

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. 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. 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.

/pdf/characterization-of-highly-efficient-heavy-ion-mutagenesis-4gs0j2jymn.pdf

Characterization of highly efficient heavy-ion mutagenesis 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 225-300keVμm(-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

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

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.

The Conflict Between Cell Proliferation and Expansion Primarily Affects Stem Organogenesis in Arabidopsis

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.

https://eprints.lib.hokudai.ac.jp/dspace/bitstream/2115/36650/1/hoshino.pdf

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea

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.

Tomonari Hirano

Papers

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

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

The Conflict Between Cell Proliferation and Expansion Primarily Affects Stem Organogenesis in Arabidopsis

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea

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