Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.     

The effectiveness of delineating ecoregions in the Kyushu region of Japan to establish environmental indicators

Throughout Japan, numerous restoration projects and efforts to conserve the river environment are underway. However, in spite of such efforts, effective measures to conserve the river ecosystem or post-evaluation of restoration projects remain insufficient due to a lack of environmental indicators. In many European countries and the United States, a number of biological indicators have been developed and adapted. However, it appears to be difficult to directly apply these indicators to Japan, because its biota is finely classified according to its many islands and peninsulas. The ultimate goal of this study is to comprehensively evaluate indicators of aquatic biodiversity from both physical and biological aspects. We divided the Kyushu region in Japan into several ecoregions as a preliminary step to establish an indicator. We delineated the ecoregions of the Kyushu region using the fish fauna data of 21 rivers within the Kyushu region. Presence–absence (0/1) data for each fish species were used to run a two-way indicator species analysis (TWINSPAN analysis). As a result, the Kyushu region was divided into four ecoregions (A: northwest Kyushu, B1: northeast Kyushu, B2: south Kyushu, and C: Amami-Oshima Island). Each ecoregion was characterized by the following fish species: (A) Cyprinidae, in particular Acheilognathinae, is abundant; (B1) Gobiidae is abundant, while Cyprinidae such as Tanakia limbata (not confirmed in B2) are also present; (B2) Gobiidae is abundant, while Rhingobius sp. DA and Rhingobius sp. CO (not confirmed in B1) are present. These results indicate that the fish fauna of the Kyushu region is finely delineated, and that an assessment standard for biological integrity should be established, based on differences in fish fauna.     

Spatially explicit neutral models for population genetics and community ecology: Extensions of the Neyman-Scott clustering process

Spatially explicit models relating to plant populations have developed little since Felsenstein (1975) pointed out that if limited seed dispersal causes clustering of individuals, such models cannot reach an equilibrium. This paper aims to resolve this issue by modifying the Neyman-Scott cluster point process. The new point processes are dynamic models with random immigration, and the continuous increase in the clustering of individuals stops at some level. Hence, an equilibrium state is achieved, and new individual-based spatially explicit neutral coalescent models are established. By fitting the spatial structure at equilibrium to individual spatial distribution data, we can indirectly estimate seed dispersal and effective population density. These estimates are improved when genetic data are available, and become even more sophisticated if spatial distribution and genetic data pertaining to the offspring are also available.     

Crown asymmetry in high latitude forests: disentangling the directional effects of tree competition and solar radiation

Light foraging by trees is a fundamental process shaping forest communities. In heterogeneous light environments this behavior is expressed as plasticity of tree growth and the development of structural asymmetries. We studied the relative influence of neighborhood structure and directional solar radiation on horizontal asymmetry of tree crowns in late‐successional high latitude (67–68°N) forests in northern Fennoscandia. We described crown asymmetries as crown vectors (i.e. horizontal vectors from stem center to crown center), which we obtained from canopy maps based on crown perimeter measurements in the field. To disentangle the influence of the two main determinants, inter‐tree competition and directionality of above‐canopy solar radiation at high latitudes, we applied circular statistical models, utilizing cylindrical distributions, to these data consisting of orientations and intensities of crown asymmetry. At the individual tree level, our model predicted crown asymmetry vectors from the current stand structure, and the predictions became better when the intensity of asymmetry (i.e. crown vector length) was higher. Competition was the main determinant of crown asymmetry for 2/3 of trees, and the model predictions improved when we incorporated the directionality of solar radiation. At the stand‐level, these asymmetries had resulted in a small increment of the projected canopy area and an increased regularity of spatial structure. Our circular statistical modelling approach provided a quantitative evaluation of the relative importance of directionality of solar radiation and neighborhood stand structure, showing how both of these factors play a role in formation of crown asymmetries in high latitude forests. This approach further demonstrated the applicability of circular statistical modeling in ecological studies where the response variable has both orientation and intensity.     

Novel assays to monitor gene expression and protein-protein interactions in rice using the bioluminescent protein,NanoLuc

Luciferases have been widely utilized as sensitive reporters to monitor gene expression and protein-protein interactions. Compared to firefly luciferase (Fluc), a recently developed luciferase, Nanoluciferase (NanoLuc or Nluc), has several superior properties such as a smaller size and stronger luminescence activity. We compared the reporter properties of Nluc and Fluc in rice (Oryza sativa). In both plant-based two-hybrid and split luc complementation (SLC) assays, Nluc activity was detected with higher sensitivity and specificity than that with Fluc. To apply Nluc to research involving the photoperiodic regulation of flowering, we made a knock-in rice plant in which the Nluc coding region was inserted in-frame with the OsMADS15 gene, a target of the rice florigen Hd3a. Strong Nluc activity in response to Hd3a, and in response to change in day length, was detected in rice protoplasts and in a single shoot apical meristem, respectively. Our results indicate that Nluc assay systems will be powerful tools to monitor gene expression and protein-protein interaction in plant research.     

Erratum to: Glutamate functions in stomatal closure in Arabidopsis and fava bean

Journal of Plant Research -     

The effects of water pollution on the phylogenetic community structure of aquatic plants in the East Tiaoxi River,China

1. Water pollution is one of the most serious aquatic environmental problems worldwide. In China, recent agricultural and industrial development has resulted in rapid changes in aquatic ecosystems. Here, we reveal the effects of water pollution on the phylogenetic community structure of aquatic macrophytes in the Tiaoxi River, China.
2. We placed a rectangular plot at 47 sites within the Tiaoxi River from the mouth of the river to 88.5 km upstream, in which we recorded species abundance and measured 22 physico-chemical variables. Bayesian phylogeny using the rbcL and matK gene sequences was employed to quantify phylogenetic α- and β-diversity, and test the phylogenetic signal in four growth forms: emergent, floating-leaved, free-floating, and submerged.
3. Within communities, water contamination and phytoplankton abundance decreased species richness and phylogenetic diversity, which resulted in phylogenetic clustering; species within communities were more closely related to each other than expected. Between communities, differences in geographical distance and phytoplankton abundance resulted in phylogenetic dissimilarity among plots. Aquatic macrophytes showed phylogenetic signals in which related species responded more similarly to disturbance.
4. Thus, the observed patterns could be explained by environmental filtering and suggested that water pollution by human activity has added more filters to the existing environmental filters that drive the species assembly of macrophyte communities.

     

Some Improvements in the Preparation of Fresh Frozen Sections

For the histochemical demonstration of sensitive enzymes it is necessary to use fresh unfixed tissue sections. With the following procedure one can constantly obtain such sections 10-20μ thick with relative ease. Schanze's sliding microtome is employed. The microtome knife is deeply cooled by placing blocks of dry ice on its surface, and is provided with a device for preventing the sections from rolling up. The microtome is operated in an ordinary refrigerator maintained at a temperature of 0-3°C. For this purpose, the door of the refrigerator is replaced by a wooden door provided with a glass window, gloved arm holes, and a small door.