Plant roots are able to sense soil nutrient availability. In order to acquire heterogeneously distributed water and minerals, they optimize their root architecture. One poorly understood plant response to soil phosphate (P(i)) deficiency is a reduction in primary root growth with an increase in the number and length of lateral roots. Here we show that physical contact of the Arabidopsis thaliana primary root tip with low-P(i) medium is necessary and sufficient to arrest root growth. We further show that loss-of-function mutations in Low Phosphate Root1 (LPR1) and its close paralog LPR2 strongly reduce this inhibition. LPR1 was previously mapped as a major quantitative trait locus (QTL); the molecular origin of this QTL is explained by the differential allelic expression of LPR1 in the root cap. These results provide strong evidence for the involvement of the root cap in sensing nutrient deficiency, responding to it, or both. LPR1 and LPR2 encode multicopper oxidases (MCOs), highlighting the essential role of MCOs for plant development.

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Root tip contact with low-phosphate media reprograms plant root architecture.

Despite advances in sequencing, the goal of obtaining a comprehensive view of genetic variation in populations is still far from reached. We sequenced 180 lines of A. thaliana from Sweden to obtain as complete a picture as possible of variation in a single region. Whereas simple polymorphisms in the unique portion of the genome are readily identified, other polymorphisms are not. The massive variation in genome size identified by flow cytometry seems largely to be due to 45S rDNA copy number variation, with lines from northern Sweden having particularly large numbers of copies. Strong selection is evident in the form of long-range linkage disequilibrium (LD), as well as in LD between nearby compensatory mutations. Many footprints of selective sweeps were found in lines from northern Sweden, and a massive global sweep was shown to have involved a 700-kb transposition.

Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden

The first step in plant breeding is to identify suitable genotypes containing the desired genes among existing varieties, or to create one if it is not found in nature. In nature, variation occurs mainly as a result of mutations and without it, plant breeding would be impossible. In this context, the major aim in mutation-based breeding is to develop and improve well-adapted plant varieties by modifying one or two major traits to increase their productivity or quality. Both physical and chemical mutagenesis is used in inducing mutations in seeds and other planting materials. Then, selection for agronomic traits is done in the first generation, whereby most mutant lines may be discarded. The agronomic traits are confirmed in the second and third generations through evident phenotypic stability, while other evaluations are carried out in the subsequent generations. Finally, only the mutant lines with desirable traits are selected as a new variety or as a parent line for cross breeding. New varieties...

https://www.tandfonline.com/doi/pdf/10.1080/13102818.2015.1087333

Principle and application of plant mutagenesis in crop improvement: a review

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An Introduction To Genetic Analysis

WRKY proteins are a large family of transcriptional regulators involved in a variety of biological processes in plants. Here we report functional characterization of a rice WRKY gene, OsWRKY89. RNA gel blot analysis indicated that OsWRKY89 was strongly induced by treatments of methyl jasmonate and UV-B radiation. The transient expression analysis of the OsWRKY89–eGFP reporter in onion epidermal cells revealed that OsWRKY89 was targeted to nuclei. Transcriptional activity assays of OsWRKY89 and its mutants fused with a GAL4 DNA binding domain indicated that the 67 C-terminal amino acids were required for the transcriptional activation and that the leucine zipper region at the N-terminus enhanced its transcriptional activity. Overexpression of OsWRKY89 led to growth retardation at the early stage and reduction of internode length. Scanning electron microscopy revealed an increase in wax deposition on leaf surfaces of the OsWRKY89 overexpression lines and a decrease in wax loading in the RNAi-mediated OsWRKY89 suppression lines. Moreover, extractable and cell-wall-bound phenolic compounds were decreased in the overexpressor lines, but its SA levels were increased. Lignin staining showed an increase in lignification in culms of the overexpressor lines. Interestingly, overexpression of the OsWRKY89 gene enhanced resistance to the rice blast fungus and white-backed planthopper as well as tolerance to UV-B irradiation. These results suggest that OsWRKY89 plays an important role in response to biotic and abiotic stresses.

Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants

An early genetic study showed that most radiation-induced mutations are not transmitted to progeny. In recent molecular studies in plants, mainly M2 plants or their progeny, which contain only transmissible mutations, have been analyzed, but the early results imply that these studies are insufficient as comprehensive descriptions of radiation-induced mutations. To study radiation-induced mutations caused by low-LET γ-rays and high-LET carbon ions at the molecular level, we used the pollen-irradiation method and the plant Arabidopsis thaliana to study various mutations, including nontransmissible mutations. This analysis revealed that most mutants induced with irradiation with γ-rays (150–600 Gy) or carbon ions (40–150 Gy) carried extremely large deletions of up to >6 Mbp, the majority of which were not transmitted to progeny. Mutations containing 1- or 4-bp deletions, which were transmitted normally, were also found. Comparison of the deleted regions in the mutants showing various manners of transmission suggests that the nontransmissibility of the large deletions may be due to the deletion of a particular region that contains a gene or genes required for gamete development or viability.

https://www.genetics.org/content/genetics/169/2/881.full.pdf

Transmissible and Nontransmissible Mutations Induced by Irradiating Arabidopsis thaliana Pollen With γ-Rays and Carbon Ions

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 ζ (AtREV3), which is thought to be involved in translesion synthesis. The rev3-1 seedlings also were sensitive to γ-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.

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Disruption of the AtREV3 Gene Causes Hypersensitivity to Ultraviolet B Light and γ-Rays in Arabidopsis: Implication of the Presence of a Translesion Synthesis Mechanism in Plants

Yokota, Y., Shikazono, N., Tanaka, A., Hase, Y., Funayama, T., Wada, S. and Inoue, M. Comparative Radiation Tolerance Based on the Induction of DNA Double-Strand Breaks in Tobacco BY-2 Cells and CHO-K1 Cells Irradiated with Gamma Rays. Radiat. Res. 163, 520–525 (2005). Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by γ rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 ± 0.1 DSBs Gbp−1 Gy−1) was only one-third of...

Comparative Radiation Tolerance Based on the Induction of DNA Double-Strand Breaks in Tobacco BY-2 Cells and CHO-K1 Cells Irradiated with Gamma Rays

Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2-derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

/pdf/an-ion-beam-induced-arabidopsis-mutant-with-marked-4zvv5max7u.pdf

An ion beam-induced Arabidopsis mutant with marked chromosomal rearrangement.

A specific gene or DNA can be directly transferred into the pollen of a plant by selectively irradiating the shell of the pollen with ion beams without affecting the cell nucleus and thereafter immersing the irradiated pollen in a solution containing the specific gene or DNA of interest. The shell of the pollen of a plant may be irradiated with ions having a linear energy transfer (LET) of 5-10,000 keV/μm to a controlled depth of ion injection. Hybridizing with pollen into which the specific gene or DNA has been transferred allows the specific gene to be transferred into a fertile embryo, thereby creating a transgenic plant.

Atsushi Tanaka

Papers

Transmissible and Nontransmissible Mutations Induced by Irradiating Arabidopsis thaliana Pollen With γ-Rays and Carbon Ions

Disruption of the AtREV3 Gene Causes Hypersensitivity to Ultraviolet B Light and γ-Rays in Arabidopsis: Implication of the Presence of a Translesion Synthesis Mechanism in Plants

Comparative Radiation Tolerance Based on the Induction of DNA Double-Strand Breaks in Tobacco BY-2 Cells and CHO-K1 Cells Irradiated with Gamma Rays

An ion beam-induced Arabidopsis mutant with marked chromosomal rearrangement.

Method of gene transfer into pollen by ion beam irradiation