Kazuhiro Shoji

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.

Nicotinate/nicotinamide mononucleotide adenyltransferase-mediated regulation of NAD biosynthesis protects guard cells from reactive oxygen species in ABA-mediated stomatal movement in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.

Cloning of a cDNA encoding an importin-α and down-regulation of the gene by light in rice leaves

Abstract The import of nuclear proteins into nuclei begins with recognition of nuclear localization signal-harboring proteins and binding to a nuclear pore targeting complex. A cDNA for an importin-α protein, a subunit of the complex, was isolated from rice plants. The amino acid sequence deduced from the nucleotide sequence of the cDNA exhibited a high homology to those of importin-α proteins from many organisms such as Arabidopsis thaliana, Saccharomyces cerevisiae, human, mouse, Xenopus laevis and Drosophila melanogaster. Down-regulation of the transcription by light was shown in the leaves of light- and dark-grown seedlings by RNA blot analysis. The down-regulation was specific to leaves, whereas no light effect was observed in root tissues or calli, in which higher levels of the transcript were detected.

Cloning of cDNA encoding the rice 22 kDa protein of Photosystem II (PSII-S) and analysis of light-induced expression of the gene

Cloning of rice cDNA encoding the chlorophyll-binding 22 kDa protein of Photosystem II (PSII-S) and the light-induced expression of the gene are reported. One of the light-responsive cDNA clones, isolated by screening with a light-specific subtracted cDNA probe, was shown to encode PSII-S of rice. Genomic Southern analysis suggested that the PSII-S gene, psbS, is a single-copy gene in rice. A brief exposure to red light induced a severalfold increase in the steady state level of PSII-S transcripts in etiolated seedlings. The red light effect was reversed by far-red light, suggesting involvement of phytochrome in the PSII-S gene regulation. Prolonged exposure (3 h) to blue light, however, revealed a much stronger effect than red light on the accumulation of PSII-S transcripts in the etiolated seedlings. In dark-adapted green plants, prolonged exposure to blue light induced re-accumulation of transcripts encoding PSII-S, whereas red light had little effect.

NAD+ Accumulation during Pollen Maturation in Arabidopsis Regulating Onset of Germination

Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.

A kinetic analysis of cadmium accumulation in a Cd hyper-accumulator fern, Athyrium yokoscense and tobacco plants

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.

Management of nitrogen fertilizer application, rather than functional gene abundance, governs nitrous oxide fluxes in hydroponics with rockwool

AimsNitrous oxide (N2O) is a strong greenhouse effective gas (GHG); the primary human source of N2O is agricultural activities. Excessive nitrogen (N) fertilization of agricultural land is now widely recognized as a major contributor. In soil, the microbial processes of nitrification and denitrification are the principal sources of N2O. However, it remains poorly understood how conventional hydroponics influences GHG emission. Here, we compared GHG fluxes from soil and rockwool used for hydroponics under identical nutrient conditions.MethodsTomato plants (Solanum lycopersicum, momotaro) were grown in soil or by hydroponics using rockwool. In situ emissions of CH4, CO2, and N2O, and the abundance of genes involved in nitrification and denitrification were measured during cultivation.ResultsHydroponics with rockwool mitigated CO2 emission by decreasing the microbial quantity in the rhizosphere. Dilution of the nutrient solution significantly decreased N2O emission from rockwool. Although proliferation of nitrifiers or denitrifiers in the rhizosphere did not induce N2O emission, reuse or long-term use of rockwool induced a 3.8-fold increase in N2O emission.ConclusionsOur data suggest that hydroponics has a lower environmental impact and that adequate fertilizer application, rather than bacterial control, governs N2O fluxes in hydroponic cultivation using rockwool.

A novel importin α from rice, a component involved in the process of nuclear protein transport1

In eukaryotes, nuclear proteins that are transported into nuclei have nuclear localization signals (NLSs), which are recognized by proteins called importin α. We isolated a rice cDNA, #61L, and the corresponding gene that encodes a protein, which shows significant homology to the importin α. Although the encoded protein had only 23–27% amino acid identity to the importin αs from various organisms including plants, the fusion protein with glutathione S‐transferase showed a specific binding activity to the NLS of SV40 T‐antigen. These results suggest that the rice #61L protein is a novel importin α in plants.

Influence of Nitrogen Limitation and Long-Term Use of Rockwool on Nitrous Oxide Emissions in Hydroponic Systems

To mitigate Nitrous Oxide (N2O) emissions derived from Nitrogen (N) fertilizer of agroecosystems, establishment of best management protocols for cultivation is necessary. Hydroponic systems using rockwool have the potential to reduce N2O emissions; however, the effects of nutrient condition and retained N compounds in rockwool on N2O emissions remain unclear. The primary objective of our study was to understand the crucial factors behind emissions of N2O. Tomato cultivation with low levels of nutrient showed reduced growth and yield, but increased N2O emission. In contrast, growth and N2O emissions were increased by cultivation with normal levels of nutrient and used (1-yold) rockwool containing excess N compounds from the previous years cultivation. Though the long-term use of rockwool significantly enhanced seasonal N2O emission, the availability of N2O precursors NO3 − and NH4 + did not clearly explain the variation in N2O fluxes during cultivation. Rather, environmental factors, such as relative water content of rockwool in the rhizosphere, were significantly correlated to N2O emissions during cultivation under various conditions. We conclude that environmental factors most strongly influence the fate of available environmental substrates remaining in rockwool, and thereby control N2O emissions.

Cascaded High-Tc Bulk Superconductor Lenses (Supertrons) for Intense Electron Beams

Two-stage tubular lenses (Supertrons) focused and transported intense electron beams (~2 kA, ~340 keV , ~10 ns) for lens separation of ~17 mm or less. In this range the second lens focused electron beams whose angle of incidence with the axis of the lens was less than the critical value of ~34°. The first and second lenses were 40 and 13 mm long, respectively, with an inner diameter of 20 mm and a wall thickness of ~1.5 mm. The lenses were made from Bi-based powder-pressed materials. Neon gas was introduced into the diode gap and into the bore of the lenses at pressures on the order of 0.1 Torr. The experimental results suggest that adjacent lenses should be positioned closer than the value of their inner diameter, and are each sufficiently long in the axial direction if longer than the value of their radii.