Rikyu Matsuki

Molecular Cloning of a Novel Importin a Homologue from Rice, by Which Constitutive Photomorphogenic 1 (COP1) Nuclear Localization Signal (NLS)-Protein Is Preferentially Nuclear Imported*

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin α, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin α homologue from rice which is in addition to our previously isolated rice importin α1a and α2, and we have named it rice importin α1b. In vitro binding and nuclear import assays using recombinant importin α1b protein demonstrate that rice importin α1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin α genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin α1b. These data suggest that each of the different rice importin α proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin α contribute to the regulation of plant nuclear protein transport.

Functional characterization of a plant importin alpha homologue. Nuclear localization signal (NLS)-selective binding and mediation of nuclear import of NLS proteins in vitro

Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin α. We recently isolated an importin α homologue from rice (rice importin α1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves. To address the function of rice importin α1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays. The rice importin α1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matα-2-type NLS, suggesting that there exists selective binding of rice importin α1 to different plant NLSs. The rice importin α1 is also capable of forming a complex with mouse importin β and NLS protein in vitro. An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin α1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data provide strong, direct evidence suggesting that rice importin α1 functions as a component of the NLS receptor in plant cells.

In vitro characterization of rice importin β1: molecular interaction with nuclear transport factors and mediation of nuclear protein import

We recently isolated two cDNAs encoding importin β homologues (rice importin β1 and β2), the first such homologues identified in plants. To address the function of rice importin β1 in the process of nuclear import of proteins, we carried out in vitro binding and nuclear import assays. Recombinant protein of rice importin β1 assembled a complex (PTAC) with rice importin α1 and NLS protein, and also bound to the nuclear envelope of tobacco BY‐2 cells. Ran‐GTP, but not Ran‐GDP, interacted with rice importin β1 and dissociated the heterodimer formed between rice importin α1 and rice importin β1. An in vitro nuclear import assay using digitonin‐permeabilized HeLa cells revealed that rice importin β1 can mediate nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data strongly suggest that rice importin β1 functions as a component of the NLS receptor in plant cells.

The Influence of Pleistocene Refugia on the Evolutionary History of the Japanese Hare, Lepus brachyurus

We performed a phylogeographic analysis of the Japanese hare, Lepus brachyurus, using the mitochondrial cytochrome b gene (1140 bp). In total, 119 haplotypes were recovered from 197 samples isolated from 82 localities on three main islands of the Japanese archipelago: Honshu, Sikoku, Kyushu, Sado Island and the Oki Islands. Results showed two distinct clades at a genetic distance of 3.5%, equivalent to an estimated 1.2 million years. The two clades, encompassing seven subclades, showed an apparent geographic affinity to Kyushu, Shikoku and the nearby area of Honshu (southern group) by one clade, whereas the other clade covered the remaining area of Honshu (northern group). The landscape shape interpolation analysis exhibited a higher genetic diversity in the southern parts of central Honshu (northern group) and Shikoku and Kyushu regions (southern group), suggesting the existence of multiple geographical origins of population expansion in each clade. The Bayesian skyline plot analysis showed that lineage diversifications occurred about 0.35, 0.20 and 0.05 million years ago (Mya), which coincide closely with the glacial—interglacial cycles during the Pleistocene. Therefore, we suggest that the Japanese hare population once inhabited northern and southern refugia, and subsequently developed several populations through local demographic fluctuations. The present day demarcation in the northern and southern geographic groups is considered to be a temporal remnant of Pleistocene population dynamics and the geographic boundary between them could move or fade away in time.

Habitat availability and density estimations for the Japanese hare by fecal pellet counting

Abstract We aimed to clarify the factors that affect Japanese hare (Lepus brachyurus) densities and habitat availability in certain vegetation types. Using fecal pellet counts, we found that hare densities and habitat availability were higher in open–tree-canopy habitats with a dense ground cover of herbs and grasses, such as cleared areas in artificial forests. However, when we examined the relationships between fecal pellets as an indicator of hare density and various attributes of forest floor vegetation, no vegetation parameters had a significant correlation with fecal pellets among different plant communities. Thus, it is difficult to predict hare density based on forest floor vegetation, in general. However, in seven Japanese cedar (Cryptomeria japonica) plantation sites, high herb volume index and a low degree of canopy closure, calculated from fisheye-lens photographs, had a significant correlation with fecal pellet density (P = 0.029 and 0.006, respectively). Based on the relationships between fecal pellets and certain forest attributes (e.g., stand height and diameter at breast height of trees), we determined that fecal pellet density was higher in young stands in these plantations. This is likely because young stands have a larger volume of herbaceous species that provide food and shelter for hares, owing to the bright ground conditions resulting from the relatively open canopy. Appropriate plantation control such as cutting and planting trees is important for habitat conservation of L. brachyurus, because planted C. japonica forest occupies more than half of artificial forests in Japan.

GPS cell phone tracking in the Greater Tokyo Area: A field test on raccoon dogs

We tested instantaneous GPS cell phone telemetry to record movement data of raccoon dogs (Nyctereutes procyonoides) in Abiko city, Greater Tokyo, Japan. We monitored the changes in locations of six raccoon dogs in various land use types such as roads, golf courses, towns, rice and crop paddies, and forest. Data were downloaded instantaneously using cell phone networks and the internet, remotely to a desktop computer. We obtained 410 location points and the maximum duration of individual tracking was 144 hours. The success rate of GPS positioning varied among individuals. On average, the success rate of three raccoon dogs in a relatively open area was 98% and that of three raccoon dogs in a mosaic area was 70%. In total, 372 of the data points (91 % of all downloaded data) with reliable accuracy were used to analyze movement speed during the day time as well as habitat use of the raccoon dogs, revealing various spatial and temporal behavioral patterns. Human activities were the likely cause of characteristic behaviors in tall weed patches and golf courses, and daily patterns of movement. Our results indicate the potential utility of GPS cell phone tracking to efficiently monitor and manage wildlife in changing urban environments.

Genetic structure of a Japanese brown frog (Rana japonica) population implies severe restriction of gene flow caused by recent urbanization in a satoyama landscape

Rapid urbanization is one of the major pressures on amphibian species. Elucidating changes in genetic structure will be useful in evaluating the effects of urbanization on amphibian populations. Our study focused on Rana japonica, which is common in complex agricultural landscapes known as satoyama, which are also under intense development pressure. We conducted landscape genetic analyses based on mitochondrial DNA haplotype frequencies of 13 breeding sites in a rapidly urbanizing area of Japan. We found several breeding sites had significantly higher F st values, and we also identified the barriers to gene flow between these sites. Observation of past aerial photographs revealed that these barriers coincided with the construction of man-made structures in the last few decades, suggesting that urbanization has restricted gene flow in R. japonica. Our results show that landscape genetic approaches are useful in conservation planning where rapid habitat degradation has taken place.

Fine-scale Genetic Structure and Estimation of Gene Flow of the Japanese Brown Frog Rana japonica in a Satoyama Landscape on the Western Side of Inba Lake, Eastern Japan

Abstract: Habitat fragmentation is one of the major threats to amphibian species. In a previous study, population genetic analyses of the Japanese brown frog Rana japonica were conducted using a mitochondrial DNA (mtDNA) marker in a typical Japanese agricultural landscape (known as satoyama) in Chiba, Japan. This previous study revealed that gene flow was restricted by the roads and cement-walled urban river that divide this site. In the present study, we reanalyzed the genetic structure of the same meta-population using microsatellite markers in comparison with the mtDNA results and elucidated fine-scale gene flow. The genetic structure derived from the microsatellite clustering analysis was almost identical to that of the mtDNA results, although some important details differed. We recognized boundaries of genetic structure are consistent with the major roads and cement-walled river, however, we also detected gene flow across those artificial barriers. We concluded that the current genetic structure was formed in the past when gene flow was strongly restricted. Gene flow among breeding populations is now being restored by the maintenance of breeding sites, although it is not sufficient to erase the signature of historical isolation.

The Rice Importin α1: Light-Regulated Expression and Mediation of Nuclear Protein Import in vitro by Recombinant Protein

Many photosynthesis- and photomorphogenesis-related genes are regulated by light. A possible regulation of light signal transduction by nuclear import processes of some transcription factor and nuclear proteins could be assumed. Phytochrome B (1) and some components of light signal transduction such as COP 1 (2) and DET1 (3) have been shown to be localized in the nuclei. Transcription factors such as GBF (4) and CCA1 (5) which bind to a regulatory cis-elements on the promoter of light responsive photosynthetic genes are also localized in the nuclei. In particular, COP1 and GBF have been reported to shuttle between nuclei and cytoplasm in response to light (2,4). These findings suggest a possible involvement of nuclear import machinery in gene regulation in plant cells.